Methods and compositions for the activation of gamma-delta T-cells

ABSTRACT

The present invention relates generally to methods and compositions for gene therapy and immunotherapy that activate gamma delta T-cells, and in particular, can be used in the treatment of various cancers and infectious diseases.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/850,937, filed on Dec. 21, 2017 entitled “Methods and Compositionsfor the Activation of Gamma-Delta T-cells” which is a continuation ofU.S. patent application Ser. No. 15/652,080, filed on Jul. 17, 2017entitled “Methods and Compositions for the Activation of Gamma-DeltaT-cells” which is a continuation of International Application No.PCT/US17/13399 filed on Jan. 13, 2017, entitled “Methods andCompositions for the Activation of Gamma-Delta T-cells” which claimspriority to U.S. Provisional Patent Application No. 62/279,474, filed onJan. 15, 2016, and entitled “Methods and Compositions for the Activationof Gamma-Delta T-cells,” the disclosures of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The present disclosure relates generally to the fields of gene therapyand immunotherapy, specifically in relation to increased activation ofgamma delta (“GD”) T cells.

BACKGROUND

Human T cells are distinguished on the basis of T cell receptorstructure. The major populations, including CD4+ and CD8+ subsets,express a receptor composed of alpha and beta chains. A smaller subsetexpresses T cell receptor made from gamma and delta chains. Gamma delta(“GD”) T cells make up 3-10% circulating lymphocytes, and Vδ2+ subsetmakes up 75% of GD T cells in blood. Vδ2+ cells recognize non-peptideepitopes and do not require antigen presentation by majorhistocompatibility complexes (“MHC”) or human leukocyte antigen (“HLA”).The majority of Vδ2+ T cells also express a Vγ9 chain and are stimulatedby exposure to 5-carbon pyrophosphate compounds that are intermediatesin mevalonate and non-mevalonate sterol/isoprenoid synthesis pathways.The response to isopentenyl pyrophosphate (5-carbon) is universal amonghealthy human beings.

Another subset of GD T cells, Vδ1+, make up a much smaller percentage ofthe T cells circulating in the blood, but Vδ+1 cells are commonly foundin the epithelial mucosa and the skin.

In general, GD T cells have several functions, including killing tumorcells and pathogen-infected cells. Stimulation through their unique Tcell receptor (“TCRs”) composed of two glycoprotein chains, γ and δ,improves the capacity for cellular cytotoxicity, cytokine secretion andother effector functions. The TCRs of GD T cells have uniquespecificities and the cells themselves occur in high clonal frequencies,thus allowing rapid innate-like responses to tumors and pathogens.

Aminobisphosphonate drugs (“ABPs”) and other inhibitors of farnesyldiphosphate synthase (“FDPS”), which are downstream from isopentenylpyrophosphate (“IPP”) in the mevalonate pathway (see, for e.g., FIG. 1),have been used to treat various diseases, including cancers,specifically those involving bone metastasis. ABPs include trade namessuch as Zometa® (Novartis) and Fosamax® (Merck).

ABPs have also been used to stimulate GD T cells. This may be becausewhen FDPS is inhibited in myeloid cells, IPP begins to accumulate andgeranylgeranyl pyrophosphate (“GGPP”), a downstream product of FDPS thatsuppresses activation of the inflammasome pathway, is reduced. Thereduction in GGPP removes an inhibitor of the caspase-dependentinflammasome pathway and allows secretion of mature cytokines includinginterleukin-beta and interleukin-18, the latter being especiallyimportant for gamma delta T cell activation.

Thus, when FDPS is blocked, the increased IPP and decreased GGPP combineto activate Vδ2+ T cells. Vδ2+ cells activated by IPP or ABPs willproliferate rapidly, express a number of cytokines and chemokines, andcan function to cytotoxically destroy tumor cells or cells infected withpathogenic microorganisms.

However, ABPs are associated with inflammation and osteonecrosis, aswell as having poor bioavailability due to their chemistry. Likewise,IPP has a very short half-life and is difficult to synthesize. Bothtypes of compounds require systemic administration in an individual.Accordingly, both ABPs in general, and IPP specifically, leave a greatdeal to be desired for therapeutic purposes.

SUMMARY OF THE INVENTION

In one aspect, a method of activating a GD T cell is provided. Themethod includes infecting, in the presence of the GD T cell, a targetcell with a viral delivery system that encodes at least one geneticelement. In embodiments, the at least one genetic element includes asmall RNA capable of inhibiting production of an enzyme involved in themevalonate pathway. In embodiments, the enzyme is FDPS. In embodiments,when the enzyme is inhibited in the target cell, the target cellsubsequently activates the GD T cell. In embodiments, the target cell isa cancer cell or a cell that has been infected with an infectious agent.In a preferred embodiment, the activation of the GD T cell results inthe GD T cell killing the cancer cell or the cell infected with aninfectious agent. In embodiments, the at least one encoded geneticelement includes a microRNA or a shRNA. In further embodiments, thetarget cell is also contacted with an aminobisphosphonate drug. Inembodiments, the aminobisphosphonate drug is zoledronic acid.

In another aspect, a method of treating cancer in a subject is provided.The method includes administering to the subject atherapeutically-effective amount of a viral delivery system that encodesat least one genetic element. In embodiments, the at least one geneticelement includes a small RNA capable of inhibiting production of anenzyme involved in the mevalonate pathway. In further embodiments, whenthe enzyme is inhibited in a cancer cell in the presence of a GD T cell,the cancer cell activates the GD T cell, to thereby treat the cancer. Inembodiments, the enzyme is FDPS. In embodiments, the at least oneencoded genetic element includes a microRNA or a shRNA. In furtherembodiments, the target cell is also contacted with anaminobisphosphonate drug. In embodiments, the aminobisphosphonate drugis zoledronic acid.

In another aspect, a method of treating an infectious disease in asubject is provided. The method includes administering to the subject atherapeutically-effective amount of a viral delivery system that encodesat least one genetic element. In embodiments, the at least one geneticelement includes a small RNA capable of inhibiting production of anenzyme involved in the mevalonate pathway. In further embodiments, whenthe enzyme is inhibited in a cell that is infected with an infectiousagent in the presence of a GD T cell, the infected cell activates the GDT cell, to thereby treat the infected cell, and the infectious disease.In embodiments, the enzyme is FDPS. In embodiments, the at least oneencoded genetic element includes a microRNA or a shRNA. In furtherembodiments, the target cell is also contacted with anaminobisphosphonate drug. In embodiments, the aminobisphosphonate drugis zoledronic acid.

In another aspect, the at least one encoded genetic element includes ashRNA having at least 80%, or at least 85%, or at least 90%, or at least95% percent identity withGTCCTGGAGTACAATGCCATTCTCGAGAATGGCATTGTACTCCAGGACTTTTT (SEQ ID NO: 1);GCAGGATTTCGTTCAGCACTTCTCGAGAAGTGCTGAACGAA ATCCTGCTTTTT (SEQ ID NO: 2);GCCATGTACATGGCAGGAATTCTCGAGAA TTCCTGCCATGTACATGGCTTTTT (SEQ ID NO: 3);or GCAGAAGGAGGCTGA GAAAGTCTCGAGACTTTCTCAGCCTCCTTCTGCTTTTT (SEQ ID NO:4). In a preferred embodiment, the shRNA includesGTCCTGGAGTACAATGCCATTCTCGAG AATGGCATTGTACTCCAGGACTTTTT (SEQ ID NO: 1);GCAGGATTTCGTTCA GCACTTCTCGAGAAGTGCTGAACGAAATCCTGCTTTTT (SEQ ID NO: 2);GCCA TGTACATGGCAGGAATTCTCGAGAATTCCTGCCATGTACATGGCTTTTT (SEQ ID NO: 3);or GCAGAAGGAGGCTGAGAAAGTCTCGAGACTTTCTCAGCCTCCTT CTGCTTTTT (SEQ ID NO:4).

In another aspect, the at least one encoded genetic element includes amicroRNA having at least 80%, or at least 85%, or at least 90%, or atleast 95% percent identity withAGGTATATTGCTGTTGACAGTGAGCGACACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGAGGCTGAGAAAGTGCTGCCTACTGCCTCGGACTTCAA GGGGCT (SEQ IDNO: 5); AAGGTATATTGCTGTTGACAGTGAGCGACACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGGCTGAGAAAGTGCTGCCTACTGCCTCGGACTTCAAGGGGCT (SEQ ID NO: 6); TGCTGTTGACAGTGAGCGACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGAGGCTGAGAAAGTTGCCTACTGCCTCGGA (SEQ ID NO: 7); CCTGGAGGCTTGCTGAAGGCTGTATGCTGACTTTCTCAGCCTCCTTCTGCTTTTGGCCACTGACTGAGCAGAAGGGCTGAGAAAGTCAGGACACAAGGCCTGTTACTAGCACTCA (SEQ ID NO: 8);CATCTCCATGGCTGTACCACCTTGTCGGGACTTTCTCAGCCTCCTTCTGCCTGTTGAATCTCATGGCAGAAGGAGGCGAGAAAGTCTGACATTTTGGTATCTTTCATCTGA CCA (SEQ ID NO:9); or GGGCCTGGCTCGAGCAGGGGGCGAGGGATACTTTCTCAGCCTCCTTCTGCTGGTCCCCTCCCCGCAGAAGGAGGCTGAGAAAGTCCTTCCCTCCCAATGACCGCGTCTTCGTCG (SEQ ID NO: 10). In a preferred embodiment, themicroRNA includes AAGGTATATTGCTGTTGACAGTGAGCGACACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGAGGCTGAGAAAGTGCTGCCTACTGCCTCGGACTTCAAGGGGCT (SEQ ID NO: 5); AAGGTATATTGCTGTTGACAGTGAGCGACACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGGCTGAGAAAGTGCTGCCTACTGCCTCGGACTTCAAGGGGCT (SEQ ID NO: 6); TGCTGTTGACAGTGAGCGACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGAGGCTGAGAAAGTTGCCTACTGCCTCGGA (SEQ ID NO: 7); CCTGGAGGCTTGCTGAAGGCTGTATGCTGACTTTCTCAGCCTCCTTCTGCTTTTGGCCACTGACTGAGCAGAAGGGCTGAGAAAGTCAGGACACAAGGCCTGTTACTAGCACTCA (SEQ ID NO: 8);CATCTCCATGGCTGTACCACCTTGTCGGGACTTTCTCAGCCTCCTTCTGCCTGTTGAATCTCATGGCAGAAGGAGGCGAGAAAGTCTGACATTTTGGTATC TTTCATCTGACCA(SEQ ID NO: 9); or GGGCCTGGCTCGAGCAGGGGGCGAGGGATACTTTCTCAGCCTCCTTCTGCTGGTCCCCTCCCCGCAGAAGGAGGCTGAGAAAGTCCTTCCCTCCCAATGACCGCGTCTTCGTCG (SEQ ID NO: 10).

In another aspect, a viral vector comprising at least one encodedgenetic element is provided. The at least one encoded genetic elementincludes a small RNA capable of inhibiting production of an enzymeinvolved in the mevalonate pathway. In embodiments, the enzyme involvedin the mevalonate pathway is famesyl diphosphate synthase (FDPS). Inembodiments, the at least one encoded genetic element includes amicroRNA or a shRNA.

In another aspect, the at least one encoded genetic element includes ashRNA having at least 80%, or at least 85%, or at least 90%, or at least95% percent identity with v In a preferred embodiment, the shRNAincludes SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; or SEQ ID NO: 4.

In another aspect, the at least one encoded genetic element includes amicroRNA having at least 80%, or at least 85%, or at least 90%, or atleast 95% percent identity with SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO:7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10. In a preferredembodiment, the microRNA includes SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO:7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10.

In embodiments, the viral vector is comprised of any vector that caneffectively transduce the small RNA into a target cell. In embodiments,the viral vector is a lentiviral vector. In other embodiments, the viralvector is an adeno-associated virus vector.

In another aspect, the viral vector includes a second encoded geneticelement. In embodiments, the second genetic element includes at leastone cytokine or chemokine. In embodiments, the at least one cytokine isselected from the group consisting of: IL-18, TNF-α, interferon-γ, IL-1,IL-2, IL-15, IL-17, and IL-12. In embodiments, the at least onechemokine is a CC chemokine or a CXC chemokine. In further embodiments,the at least one chemokine is RANTES.

In another aspect, a lentiviral vector system for expressing alentiviral particle is provided. The system includes a lentiviralvector, at least one envelope plasmid for expressing an envelope proteinoptimized for infecting a cell; and at least one helper plasmid forexpressing gag, pol, and rev genes. When the lentiviral vector, the atleast one envelope plasmid, and the at least one helper plasmid aretransfected into a packaging cell, a lentiviral particle is produced bythe packaging cell. In embodiments, the lentiviral particle is capableof infecting a targeting cell, and inhibiting an enzyme involved in themevalonate pathway within the target cell. In embodiments, the enzymeinvolved in the mevalonate pathway is FDPS. In embodiments, thelentiviral vector system includes a first helper plasmid for expressingthe gag and pol genes, and a second helper plasmid for expressing therev gene. In embodiments, the envelope protein is preferably optimizedfor infecting a target cell. In embodiments, the target cell is a cancercell. In other embodiments, the target cell is a cell that is infectedwith an infectious agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an overview of the major steps in the mevalonate pathwayfor biosynthesis of steroids and isoprenoids.

FIG. 2 depicts an exemplary 3-vector lentiviral vector system in acircularized form.

FIG. 3 depicts an exemplary 4-vector lentiviral vector system in acircularized form.

FIG. 4 depicts: (A) a linear map of a lentiviral vector expressing aFDPS shRNA targeting sequence; and (B) a linear map of a lentiviralvector expressing a synthetic microRNA with a FDPS targeting sequence.

FIG. 5 depicts data demonstrating activation of Vδ2+ T cells THP-1leukemia cells with a lentivirus expressing FDPS shRNA #4 (SEQ ID NO:4), as described herein.

FIG. 6 depicts data demonstrating activation of Vδ2+ T cells by THP-1leukemia cells with a lentivirus expressing FDPS shRNA #4 (SEQ ID NO:4), as described herein.

FIG. 7 depicts data demonstrating activation of Vδ2+ T cells by PC3prostate carcinoma cells with a lentivirus expressing FDPS shRNA #1 (SEQID NO: 1), as described herein.

FIG. 8 depicts data demonstrating activation of Vδ2+ T cells by PC3prostate carcinoma cells with a lentivirus expressing FDPS shRNA #4 (SEQID NO: 4), as described herein.

FIG. 9 depicts data demonstrating activation of Vδ2+ T cells by HepG2carcinoma cells with a lentivirus expressing FDPS shRNA #1 (SEQ IDNO: 1) or FDPS shRNA #4 (SEQ ID NO: 4), as described herein.

FIG. 10 depicts data demonstrating activation of Vδ2+ T cells by THP-1leukemia cells with a lentivirus expressing miR30 FDPS #1 (SEQ ID NO:5), as described herein.

FIG. 11 depicts data demonstrating the percent of specific lysis versusan E:T ratio for a variety of experimental conditions, as describedherein.

FIG. 12 depicts data demonstrating lentiviral-delivered shRNA-based RNAinterference targeting the human FDPS gene.

FIG. 13 depicts data demonstrating lentiviral-delivered miR-based RNAinterference targeting the human FDPS gene.

FIG. 14 depicts data demonstrating activation of Vδ2+ T cells by HepG2carcinoma cells with an adeno-associated virus expressing FDPS shRNA #4(SEQ ID NO: 4), as described herein.

FIG. 15 depicts immunoblot data demonstrating lack of RAP1 prenylationin the cells transduced with LV-shFDPS and treated with zoledronic acid.

DETAILED DESCRIPTION Overview of Disclosure

The present disclosure relates to gene therapy constructs and deliveryof the same to cells, resulting in suppression of Famesyl diphosphatesynthase (“FDPS”), which is necessary to convert isopentenyl phosphate(IPP) to famesyl diphosphate (FDP), as shown, for example, in FIG. 1. Inembodiments, one or more viral vectors are provided with microRNAs orshort homology RNAs (shRNA) that target FDPS, thereby reducingexpression levels of this enzyme. The viral vectors include lentiviralvectors and AAV vectors. A consequence of modulating expression of FDPSis to increase the accumulation of IPP, which is a stimulator of GD Tcell proliferation and differentiation. Accordingly, the constructsprovided herein are used to activate GD T cells, and are used to treatcancers and infectious diseases.

Definitions and Interpretation

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclature used in connection with, and techniques of, cell and tissueculture, molecular biology, immunology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well-known and commonly used in the art. The methods andtechniques of the present disclosure are generally performed accordingto conventional methods well-known in the art and as described invarious general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g.: Sambrook J. & Russell D. Molecular Cloning: ALaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2000); Ausubel et al., Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, Wiley, John & Sons, Inc. (2002); Harlow and Lane UsingAntibodies: A Laboratory Manual; Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols inProtein Science, Wiley, John & Sons, Inc. (2003). Any enzymaticreactions or purification techniques are performed according tomanufacturer's specifications, as commonly accomplished in the art or asdescribed herein. The nomenclature used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well-known and commonly used in the art.

As used in the description and the appended claims, the singular forms“a”, “an” and “the” are used interchangeably and intended to include theplural forms as well and fall within each meaning, unless the contextclearly indicates otherwise. Also, as used herein, “and/or” refers toand encompasses any and all possible combinations of one or more of thelisted items, as well as the lack of combinations when interpreted inthe alternative (“or”).

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1. It is to be understood, althoughnot always explicitly stated that all numerical designations arepreceded by the term “about”. The term “about” also includes the exactvalue “X” in addition to minor increments of “X” such as “X+0.1” or“X−0.1.” It also is to be understood, although not always explicitlystated, that the reagents described herein are merely exemplary and thatequivalents of such are known in the art.

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent depending uponthe context in which it is used. If there are uses of the term which arenot clear to persons of ordinary skill in the art given the context inwhich it is used, “about” will mean up to plus or minus 10% of theparticular term.

The terms “administration of” or “administering” an active agent shouldbe understood to mean providing an active agent to the subject in needof treatment in a form that can be introduced into that individual'sbody in a therapeutically useful form and therapeutically effectiveamount.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the composition or method. “Consisting of” shall meanexcluding more than trace elements of other ingredients for claimedcompositions and substantial method steps. Embodiments defined by eachof these transition terms are within the scope of this disclosure.Accordingly, it is intended that the methods and compositions caninclude additional steps and components (comprising) or alternativelyincluding steps and compositions of no significance (consistingessentially of) or alternatively, intending only the stated method stepsor compositions (consisting of).

As used herein, “expression,” “expressed,” or “encodes” refers to theprocess by which polynucleotides are transcribed into mRNA and/or theprocess by which the transcribed mRNA is subsequently being translatedinto peptides, polypeptides, or proteins. Expression may includesplicing of the mRNA in a eukaryotic cell or other forms ofpost-transcriptional modification or post-translational modification.

The term “famesyl diphosphate synthase” may also be referred to hereinas FDPS, and may also be referred to herein as famesyl pyrophosphatesynthase or FPPS.

The term “gamma delta T cell” may also be referred to herein as a γδ Tcell, or further as a GD T cell. The term “gamma delta T cellactivation” refers to any measurable biological phenomenon associatedwith a gamma delta T cell that is representative of such T cell beingactivated. Non-limiting examples of such a biological phenomenon includean increase of cytokine production, changes in the qualitative orquantitative composition of cell surface proteins, an increase in T cellproliferation, and/or an increase in T cell effector function, suchkilling or a target cell or assisting another effector cell to kill atarget cell.

The terms “individual,” “subject,” and “patient” are usedinterchangeably herein, and refer to any individual mammal subject,e.g., bovine, canine, feline, equine, or human.

The term “miRNA” refers to a microRNA, and also may be referred toherein as “miR”.

The term “packaging cell line” refers to any cell line that can be usedto express a lentiviral particle.

The term “percent identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, the“percent identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared. For sequencecomparison, typically one sequence acts as a reference sequence to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsobe determined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present disclosure canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acidmolecules provided in the disclosure. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3 to obtainamino acid sequences homologous to the protein molecules of thedisclosure. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

As used herein, “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissues,organs, and/or bodily fluids of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problems orcomplications commensurate with a reasonable benefit/risk ratio.

As used herein, a “pharmaceutically acceptable carrier” refers to, andincludes, any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Thecompositions can include a pharmaceutically acceptable salt, e.g., anacid addition salt or a base addition salt (see, e.g., Berge et al.(1977) J Pharm Sci 66:1-19).

As used herein, the term “SEQ ID NO” is synonymous with the term“Sequence ID No.”

As used herein, “small RNA” refers to non-coding RNA that are generallyless than about 200 nucleotides or less in length and possess asilencing or interference function. In other embodiments, the small RNAis about 175 nucleotides or less, about 150 nucleotides or less, about125 nucleotides or less, about 100 nucleotides or less, or about 75nucleotides or less in length. Such RNAs include microRNA (miRNA), smallinterfering RNA (siRNA), double stranded RNA (dsRNA), and short hairpinRNA (shRNA). “Small RNA” of the disclosure should be capable ofinhibiting or knocking-down gene expression of a target gene, generallythrough pathways that result in the destruction of the target gene mRNA.

The term “therapeutically effective amount” refers to a sufficientquantity of the active agents of the present disclosure, in a suitablecomposition, and in a suitable dosage form to treat or prevent thesymptoms, progression, or onset of the complications seen in patientssuffering from a given ailment, injury, disease, or condition. Thetherapeutically effective amount will vary depending on the state of thepatient's condition or its severity, and the age, weight, etc., of thesubject to be treated. A therapeutically effective amount can vary,depending on any of a number of factors, including, e.g., the route ofadministration, the condition of the subject, as well as other factorsunderstood by those in the art.

As used herein, the term “therapeutic vector” includes, withoutlimitation, reference to a lentiviral vector or an AAV vector.

“A treatment” is intended to target the disease state and combat it,i.e., ameliorate or prevent the disease state. The particular treatmentthus will depend on the disease state to be targeted and the current orfuture state of medicinal therapies and therapeutic approaches. Atreatment may have associated toxicities.

The term “treatment” or “treating” generally refers to an interventionin an attempt to alter the natural course of the subject being treated,and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects include, but are not limited to,preventing occurrence or recurrence of disease, alleviating symptoms,suppressing, diminishing or inhibiting any direct or indirectpathological consequences of the disease, ameliorating or palliating thedisease state, and causing remission or improved prognosis.

DESCRIPTION OF ASPECTS OF THE DISCLOSURE

In one aspect, a method of activating a GDT cell is provided. The methodincludes infecting, in the presence of the GD T cell, a target cell witha viral delivery system encoding at least one genetic element. Inembodiments, the at least one encoded genetic element includes a smallRNA capable of inhibiting production of an enzyme involved in themevalonate pathway. In embodiments, the enzyme is FDPS. In embodiments,when the enzyme is inhibited in the target cell, the target cellactivates the GD T cell. In embodiments, the target cell is a cancercell or a cell that has been infected with an infectious agent. Inembodiments, the at least one encoded genetic element includes amicroRNA or a shRNA.

In embodiments, the at least one encoded genetic element includes ashRNA having at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95% or more percent identity withGTCCTGGAGTACAATGCCATTCTCGAGAATGGCATTGTACTCCAGGACTTTTT (SEQ ID NO: 1);GCAGGATTTCGTTCAGCACTTCTCGAGAAGTGCTGAACGAA ATCCTGCTTTTT (SEQ ID NO: 2);GCCATGTACATGGCAGGAATTCTCGAGAA TTCCTGCCATGTACATGGCTTTTT (SEQ ID NO: 3);or GCAGAAGGAGGCTGA GAAAGTCTCGAGACTTTCTCAGCCTCCTTCTGCTTTTT (SEQ ID NO:4). In a preferred embodiment, the shRNA includesGTCCTGGAGTACAATGCCATTCTCGAG AATGGCATTGTACTCCAGGACTTTTT (SEQ ID NO: 1);GCAGGATTTCGTTCA GCACTTCTCGAGAAGTGCTGAACGAAATCCTGCTTTTT (SEQ ID NO: 2);GCCA TGTACATGGCAGGAATTCTCGAGAATTCCTGCCATGTACATGGCTTTTT (SEQ ID NO: 3);or GCAGAAGGAGGCTGAGAAAGTCTCGAGACTTTCTCAGCCTCCTT CTGCTTTTT (SEQ ID NO:4).

In another aspect, the at least one encoded genetic element includes amicroRNA having at least 80%, at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95% or more percent identity withAAGGTATATTGCTGTTGACAGTGAGCGACACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGAGGCTGAGAAAGTGCTGCCTACTGCCTCGGACTTCA AGGGGCT (SEQ IDNO: 5); AAGGTATATTGCTGTTGACAGTGAGCGACACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGGCTGAGAAAGTGCTGCCTACTGCCTCGGACTTCAAGGGGCT (SEQ ID NO: 6); TGCTGTTGACAGTGAGCGACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGAGGCTGAGAAAGTTGCCTACTGCCTCGGA (SEQ ID NO: 7); CCTGGAGGCTTGCTGAAGGCTGTATGCTGACTTTCTCAGCCTCCTTCTGCTTTTGGCCACTGACTGAGCAGAAGGGCTGAGAAAGTCAGGACACAAGGCCTGTTACTAGCACTCA (SEQ ID NO: 8);CATCTCCATGGCTGTACCACCTTGTCGGGACTTTCTCAGCCTCCTTCTGCCTGTTGAATCTCATGGCAGAAGGAGGCGAGAAAGTCTGACATTTTGGTATCTTTCATCTGA CCA (SEQ ID NO:9); or GGGCCTGGCTCGAGCAGGGGGCGAGGGATACTTTCTCAGCCTCCTTCTGCTGGTCCCCTCCCCGCAGAAGGAGGCTGAGAAAGTCCTTCCCTCCCAATGACCGCGTCTTCGTCG (SEQ ID NO: 10). In a preferred embodiment, themicroRNA includes AAGGTATATTGCTGTTGACAGTGAGCGACACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGAGGCTGAGAAAGTGCTGCCTACTGCCTCGGACTTCAAGGGGCT (SEQ ID NO: 5); AAGGTATATTGCTGTTGACAGTGAGCGACACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGGCTGAGAAAGTGCTGCCTACTGCCTCGGACTTCAAGGGGCT (SEQ ID NO: 6); TGCTGTTGACAGTGAGCGACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGAGGCTGAGAAAGTTGCCTACTGCCTCGGA (SEQ ID NO: 7); CCTGGAGGCTTGCTGAAGGCTGTATGCTGACTTTCTCAGCCTCCTTCTGCTTTTGGCCACTGACTGAGCAGAAGGGCTGAGAAAGTCAGGACACAAGGCCTGTTACTAGCACTCA (SEQ ID NO: 8);CATCTCCATGGCTGTACCACCTTGTCGGGACTTTCTCAGCCTCCTTCTGCCTGTTGAATCTCATGGCAGAAGGAGGCGAGAAAGTCTGACATTTTGGTATC TTTCATCTGACCA(SEQ ID NO: 9); or GGGCCTGGCTCGAGCAGGGGGCGAGGGATACTTTCTCAGCCTCCTTCTGCTGGTCCCCTCCCGCAGAAGGAGGCTGAGAAAGTCCTTCCCTCCCAATGACCGCGTCTTCGTCG (SEQ ID NO: 10).

In another aspect, the target cell is also contacted with anaminobisphosphonate drug. In a preferred embodiment, theaminobisphosphonate drug is zoledronic acid.

In another aspect, a method of treating cancer in a subject is provided.The method includes administering to the subject atherapeutically-effective amount of a viral delivery system encoding atleast one genetic element. In embodiments, the at least one encodedgenetic element includes a small RNA capable of inhibiting production ofan enzyme involved in the mevalonate pathway. In further embodiments,when the enzyme is inhibited in a cancer cell in the presence of a GD Tcell, the cancer cell activates the GD T cell, to thereby treat thecancer. In embodiments, the enzyme is FDPS. In embodiments, the at leastone encoded genetic element includes a microRNA or a shRNA.

In another aspect, a method of treating an infectious disease in asubject is provided. The method includes administering to the subject atherapeutically-effective amount of a viral delivery system encoding atleast one genetic element. In embodiments, the at least one encodedgenetic element includes a small RNA capable of inhibiting production ofan enzyme involved in the mevalonate pathway. In further embodiments,when the enzyme is inhibited in a cell that is infected with aninfectious agent and is in the presence of a GD T cell, the infectedcell activates the GD T cell, to thereby treat the infected cell, andthe infectious disease. In embodiments, the enzyme is FDPS. Inembodiments, the at least one encoded genetic element includes amicroRNA or a shRNA.

In embodiments, the at least one encoded genetic element includes ashRNA having at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95% or more percent identity with SEQ ID NO: 1; SEQID NO: 2; SEQ ID NO: 3; or SEQ ID NO: 4. In a preferred embodiment, theshRNA includes SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; or SEQ ID NO:4.

In other embodiments, the at least one encoded genetic element includesa microRNA having at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95% or more percent identity with SEQ ID NO:5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO:10. In a preferred embodiment, the microRNA includes SEQ ID NO: 5; SEQID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10.

In another aspect, a viral vector comprising at least one encodedgenetic element is provided. The at least one encoded genetic elementincludes a small RNA capable of inhibiting production of an enzymeinvolved in the mevalonate pathway. In embodiments, the enzyme involvedin the mevalonate pathway is famesyl diphosphate synthase (FDPS). Inembodiments, the at least one encoded genetic element includes amicroRNA or a shRNA.

In another aspect, the at least one encoded genetic element includes ashRNA having at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95% or more percent identity with SEQ ID NO: 1; SEQID NO: 2; SEQ ID NO: 3; or SEQ ID NO: 4. In a preferred embodiment, theshRNA includes SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; or SEQ ID NO:4.

In another aspect, the at least one encoded genetic element includes amicroRNA having at least 80%, at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95% or more percent identity with SEQ ID NO: 5; SEQID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10. Ina preferred embodiment, the microRNA includes SEQ ID NO: 5; SEQ ID NO:6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10.

In embodiments, the viral vector includes any vector that caneffectively transduce the small RNA. In embodiments, the viral vector isa lentiviral vector. In other embodiments, the viral vector is anadeno-associated virus (AAV) vector.

In another aspect, the viral vector includes a second encoded geneticelement. In embodiments, the second genetic element includes at leastone cytokine or chemokine. In embodiments, the at least one cytokine isselected from the group consisting of: IL-18, TNF-α, interferon-γ, IL-1,IL-2, IL-15, IL-17, and IL-12. In embodiments, the at least onechemokine is a CC chemokine, CXC chemokine, c CX3 chemokine or a XCchemokine. In a further embodiment, the at least one chemokine is the CCchemokine, RANTES.

In another aspect, a lentiviral vector system for expressing alentiviral particle is provided. The system includes a lentiviralvector, at least one envelope plasmid for expressing an envelope proteinoptimized for infecting a cell; and at least one helper plasmid forexpressing gag, pol, and rev genes. When the lentiviral vector, the atleast one envelope plasmid, and the at least one helper plasmid aretransfected into a packaging cell, a lentiviral particle is produced bythe packaging cell. In embodiments, the lentiviral particle is capableof infecting a targeting cell, and inhibiting an enzyme involved in themevalonate pathway within the target cell. In embodiments, the enzymeinvolved in the mevalonate pathway is FDPS. In embodiments, thelentiviral vector system includes a first helper plasmid for expressingthe gag and pol genes, and a second helper plasmid for expressing therev gene. In embodiments, the envelope protein is preferably optimizedfor infecting a target cell. In embodiments, the target cell is a cancercell. In other embodiments, the target cell is a cell that is infectedwith an infectious disease.

Cancer

The compositions and methods provided herein are used to treat cancer. Acell, tissue, or target may be a cancer cell, a cancerous tissue, harborcancerous tissue, or be a subject or patient diagnosed or at risk ofdeveloping a disease or condition. In certain aspects, a cell may be anepithelial, an endothelial, a mesothelial, a glial, a stromal, or amucosal cell. The cancer cell population can include, but is not limitedto a brain, a neuronal, a blood, an endometrial, a meninges, anesophageal, a lung, a cardiovascular, a liver, a lymphoid, a breast, abone, a connective tissue, a fat, a retinal, a thyroid, a glandular, anadrenal, a pancreatic, a stomach, an intestinal, a kidney, a bladder, acolon, a prostate, a uterine, an ovarian, a cervical, a testicular, asplenic, a skin, a smooth muscle, a cardiac muscle, or a striated musclecell, can also include a cancer cell population from any of theforegoing, and can be associated with one or more of carcinomas,sarcomas, myelomas, leukemias, lymphomas, mixed types or mixtures of theforegoing. In still a further aspect cancer includes, but is not limitedto astrocytoma, acute myeloid leukemia, anaplastic large cell lymphoma,acute lymphoblastic leukemia, angiosarcoma, B-cell lymphoma, Burkitt'slymphoma, breast carcinoma, bladder carcinoma, carcinoma of the head andneck, cervical carcinoma, chronic lymphoblastic leukemia, chronicmyeloid leukemia, colorectal carcinoma, endometrial carcinoma,esophageal squamous cell carcinoma, Ewing's sarcoma, fibrosarcoma,glioma, glioblastoma, gastrinoma, gastric carcinoma, hepatoblastoma,hepatocellular carcinoma, Kaposi's sarcoma, Hodgkin lymphoma, laryngealsquamous cell carcinoma, larynx carcinoma, leukemia, leiomyosarcoma,lipoma, liposarcoma, melanoma, mantle cell lymphoma, medulloblastoma,mesothelioma, myxofibrosarcoma, myeloid leukemia, mucosa-associatedlymphoid tissue B cell lymphoma, multiple myeloma, high-riskmyelodysplastic syndrome, nasopharyngeal carcinoma, neuroblastoma,neurofibroma, high-grade non-Hodgkin lymphoma, non-Hodgkin lymphoma,lung carcinoma, non-small cell lung carcinoma, ovarian carcinoma,oesophageal carcinoma, osteosarcoma, pancreatic carcinoma,pheochromocytoma, prostate carcinoma, renal cell carcinoma,retinoblastoma, rhabdomyosarcoma, salivary gland tumor, Schwanomma,small cell lung cancer, squamous cell carcinoma of the head and neck,testicular tumor, thyroid carcinoma, urothelial carcinoma, and Wilm'stumor.

The compositions and methods provided herein are also used to treatNSCLC (non-small cell lung cancer), pediatric malignancies, cervical andother tumors caused or promoted by human papilloma virus (HPV),melanoma, Barrett's esophagus (pre-malignant syndrome), adrenal and skincancers and auto immune, neoplastic cutaneous diseases.

Infectious Diseases

The compositions and methods disclosed herein can be used to treatinfectious diseases. The term “infectious disease” includes any diseasethat is caused by an infectious agent. An “infectious agent” includesany exogenous pathogen including, without limitation, bacteria, fungi,viruses, mycoplasma, and parasites. Infectious agents that may betreated with compositions provided for in this disclosure include anyart-recognized infectious organisms that cause pathogenesis in ananimal, including such organisms as bacteria that are gram-negative orgram-positive cocci or bacilli, DNA and RNA viruses, including, but notlimited to, DNA viruses such as papilloma viruses, parvoviruses,adenoviruses, herpesviruses and vaccinia viruses, and RNA viruses, suchas arenaviruses, coronaviruses, rhinoviruses, respiratory syncytialviruses, influenza viruses, picomaviruses, paramyxoviruses, reoviruses,retroviruses, and rhabdoviruses. Examples of fungi that may be treatedwith the compositions and methods of the disclosure include fungi thatgrow as molds or are yeastlike, including, for example, fungi that causediseases such as ringworm, histoplasmosis, blastomycosis, aspergillosis,cryptococcosis, sporotrichosis, coccidioidomycosis,paracoccidio-idomycosis, and candidiasis. Compositions and methodsprovided for herein may be utilized to treat parasitic infectionsincluding, but not limited to, infections caused by somatic tapeworms,blood flukes, tissue roundworms, ameba, and Plasmodium, Trypanosoma,Leishmania, and Toxoplasma species.

Methods of GD T Cell Activation

Provided herein are compositions and methods for activating GD T cellsin an individual, as well as methods for treating tumors and infectiousdiseases. For instance, in embodiments, the compositions and methodsprovided herein can be used in methods to treat all known cancersbecause activated GD T cells comprise a natural mechanism for immunesurveillance of tumors (See for e.g.: Pauza et al. 2014 Frontiers inImmunol. 5:687). Likewise, in embodiments, the compositions and methodsprovided herein can be used to treat infectious diseases, including butnot limited to flavivirus, influenza virus, human retrovirus,mycobacteria, plasmodia and a variety of other viral, fungal andbacterial infections. (See for e.g.: Pauza and Cairo, 2015 Cell Immunol.296(1).

In general, a vector system is administered to an individual totransfect or transduce a target cell population with the disclosedconstructs for decreasing expression of FDPS and, in other embodiments,increasing expression of chemokines or cytokines. Administration andtransfection/transduction can occur in vivo or ex vivo, with thetransfected cells later administered back into the subject in the latterscenario.

Administration of the disclosed vectors and transfection or transductionof the disclosed constructs into a subject's cells result in decreasedexpression of FDPS, increased expression of cytokines or chemokines,accumulation of IPP and in many cases, reduced growth rates forgenetically modified tumor cells. All of these features work together toactivate and co-localize GD T cells to the site of a tumor or infection.

The disclosed methods can also increase the capacity of NK cells torecognize and destroy tumor cells and/or infected cells. Crosstalkbetween GD T cells and NK cells is an important aspect of regulating theimmune and inflammatory responses. Further, GD T cells are known totrigger dendritic cell maturation, recruit B cells and macrophages, andparticipate in a variety of cytolytic activities, such as secretion ofinterferon-γ and TNF-α.

In embodiments, the disclosed compositions and methods provided hereincomprise a form of gene therapy for activating GD T cells at the site oftumor or infectious disease pathology. In an aspect, the compositionsand methods provided herein activate GD T cells and support theirproliferation, differentiation, and functional capacities by promotingthe production of specific cytokines needed for cytolytic activitycapable of killing cancer cells or treating infectious diseases.

In embodiments the gene therapy sequences (e.g., FDPS shRNAs) arecarried by therapeutic vectors, including but not limited to viralvectors such as lentiviruses or adeno-associated viruses, although otherviral vectors can also be suitable. Gene therapy constructs may also bedelivered in the form of DNA or RNA, including but not limited toplasmid forms. In embodiments, the disclosed gene therapy constructs mayalso be delivered in the form of protein-nucleic acid complexes or lipidnucleic acid complexes and mixtures of these formulations. For instance,a protein-nucleic acid complex can comprise nucleic acids of interest ina complex with cationic peptides such as lysine and arginine.Lipid-nucleic acids complexes can comprise lipid emulsions, micelles,liposomes, and/or mixtures of neutral and cationic lipids such as DOTMA,DOSPA, DOTAP, and DMRIE.

In embodiments, therapeutic vectors may comprise a single construct orat least two, at least three, at least four, or at least five differentconstructs. When more than one construct is present in a vector theconstructs may be identical, or they may be different. For instance, theconstructs may vary in terms of their promoters, the presence or absenceof an integrating elements, and/or their sequences. In some embodiments,a therapeutic vector will comprise at least one construct that encodes asmall RNA capable of knocking down the expression of FDPS. Inembodiments, the therapeutic vector will also encode a specificcytokine(s) and/or chemokine(s), including but not limited to TNF-α,interferon-γ, IL-1, IL-2, IL-15, IL-17, IL-18 or IL-12. In someembodiments, a single construct may encode both small RNAs capable ofknocking down the expression of FDPS and specific cytokines orchemokines, including but not limited to TNF-α, interferon-γ, IL-1,IL-2, IL-15, IL-17, IL-18 or IL-12.

In embodiments, viral vectors may introduce nucleic acid constructs thatbecome integrated into the host chromosome. Alternately, transientdelivery vectors may be used to prevent chromosomal integration andlimit the lifespan of gene therapy constructs.

In embodiments, the disclosed constructs and vectors comprise shorthomology region RNA (“shRNA”), micro RNA (“miRNA”), or siRNA capable ofreducing or knocking down expression of FDPS and/or geranylpyrophosphate synthase (“GPPS”) and/or famesyl transferase (“FT”) genes.By down regulating these genes, which control steroid and isoprenoidsynthesis, isopentenyl pyrophosphate (“IPP”) levels are elevated.Elevation and accumulation of IPP is a known mechanism for increasing GDT cells activation. Further, down regulation of these pyrophosphatesynthase genes removes an important negative regulator of inflammasomefunction that in turn results in increased expression of cytokines thatare important for GD T cell activation and effector cell function.

In embodiments, the disclosed constructs are regulated by specificpromoters that are capable of producing interleukin-2 and/orinterleukin-15 to sustain GD T cell proliferation. In addition, thedisclosed constructs may be regulated by specific promoters that arecapable of producing interleukin-1 beta and/or interleukin-18 and/orinterferon-gamma required for GD T cell differentiation and acquisitionof all effector cell function. Desirable effector cell functions includethe capacity for direct cytotoxic cell killing of tumors and/or infectedcells, secretion of beneficial cytokines and/or chemokines, increasedexpression of NK receptors required to recognize cancerous or infectedcells, and increased expression of Fc receptors needed to bind targetingantibodies in order to co-localize GD T cells with cancerous or infectedcell targets.

In embodiments, the disclosed methods activate GD T cells, resulting inthe indirect effect of increasing the capacity for NK cells to attackand destroy cancerous cells, tumors, or infected cells. The activationof NK cells requires GD T cells that are stimulated to proliferate anddifferentiate, and to express 4-1BBL costimulatory ligand needed toengage the 4-1BB costimulatory receptor on NK cells. This form ofcrosstalk is known as an important mechanism for activating NK cells andis achieved here through the action of the disclosed methods andcompositions.

In another aspect, crosstalk between GD T cells and NK cells is animportant mechanism for eliminating inflammatory dendritic cells thataccumulate in diseased tissues. Alone, neither GD T cells nor NK cellsare capable of destroying dendritic cells, but once the aforementionedcrosstalk interactions have occurred, NK cells are altered to becomecytotoxic against inflammatory dendritic cells. This immuno-regulatorymechanism depends on strong activation and proliferation of GD T cells.

In embodiments, the disclosed methods for activation of GD T cellsfurther comprise a step of suppressing pathologic inflammatory responsesthat may include cellular proliferation leading to atherosclerosis,chronic immune activation that stimulates tumor growth, autoimmunediseases including psoriasis and other presentations in the epidermis,inflammatory diseases of the central nervous system, and arthritis andother diseases of unregulated immune responses.

In embodiments, therapeutic vectors are administered concurrently withaminobisphosphonate (ABP) drugs to achieve synergistic activation ofgamma delta T cells. The synergism can allow alternate, modified orreduced doses of ABP and may decrease adverse reactions to ABP includingacute inflammatory responses and chronic diseases.

Constructs for GD T Cell Activation

Inhibition of FDPS results in IPP accumulation, resulting in activationof Vδ2+GD T cells and expression of IL-18, which is also important inactivating GD T cells. Inhibition of famesyl transferase results indecreased prenylation of proteins. The disclosed constructs can betransfected or transduced into specific target cells, like tumor cellsor infected cells, where they can express RNA sequences (i.e., siRNA,shRNA or microRNA) that will inhibit translation of FDPS as well asencode and express cytotoxic cytokines or chemokines.

Disclosed herein are constructs for decreasing expression of FDPS and/orFT, increasing expression of cytokines, and increasing expression ofchemokines including RANTES. For instance, in some embodiments theconstructs may encode for interferon-gamma, IL-1, IL-2, IL-15, IL-17,IL-18 or IL-12.

Expression of cytokines and chemokines, like those listed above, willresult in localized cytotoxic destruction of tumor cells or cellsinfected with pathogenic organisms. Accordingly, expression of suchconstructs by a tumor cell or an infected cell will result in theunwanted cells assisting in its own destruction.

Likewise, if the disclosed constructs are expressed in a tumor cell orinfected cell, decreasing the expression of FDPS and FT will result inactivation and recruitment of GD T cells to the tumor site of site ofcell infection. Increasing expression of RANTES will further attract GDT cells to intended tissue location. Because GD T cells can kill a broadrange of tumors of epithelial origin as well as many leukemias andlymphomas, and are further able to produce high levels of the anti-tumorcytokine, IFNγ, recruitment of GD T cells to the site of a tumor can bea particularly effective means of inducing anti-tumor immunity.

Decreased expression of FDPS can be achieved via shRNA, microRNA, siRNA,or other means known in the art. For instance, shRNAs according to SEQID NOS: 1, 2, 3, or 4, or variants thereof can be used in the disclosedconstructs and methods, although this example is not limiting. Thecoding regions for RNAs to decrease expression of FDPS and FT and thecoding regions of cytokine and chemokines may be in the same constructor on different constructs.

The classical approach for the production of recombinant polypeptides orgene regulatory molecules including small RNA is the use of stableexpression constructs. These constructs are based upon chromosomalintegration of a transduced expression plasmid (or at least a portionthereof) into the genome of the host cell, short-duration plasmidtransfection, or non-integrating viral vectors also with limitedhalf-life. The sites of gene integration are generally random, and thenumber and ratio of genes integrating at any particular site are oftenunpredictable; likewise, non-integrating plasmids or viral vectors alsogenerate nuclear DNA but these species usually lack sequences requiredfor DNA replication and continuous maintenance. Thus, constructs thatrely on chromosomal integration result in permanent maintenance of therecombinant gene that may exceed the therapeutic interval.

An alternative to stable expression constructs for gene expression aretransient expression constructs. The expression of the latter geneexpression construct is based on non-integrated plasmids, and hence theexpression is typically lost as the cell undergoes division or theplasmid vectors are destroyed by endogenous nucleases.

The disclosed constructs are preferably episomal constructs that aretransiently expressed. Episomal constructs are degraded or diluted overtime such that they do not make permanent changes to a subject's genome,nor are they incorporated into the chromosome of a target cell. Theprocess of episomal replication typically incorporates both host cellreplication machinery and viral trans-acting factors.

Avoiding chromosomal integration reduces certain barriers to in vivogene delivery. However, even integration-defective constructs can have abackground frequency of integration, and any DNA molecule can find rarehomologies to recombine with host sequences; but these rates ofintegration are exceptionally rare and generally not clinicallysignificant.

Thus, in some embodiments, the disclosed vectors support active geneand/or small RNA delivery over a period of about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, or about 12 weeks. In some embodiments, the disclosed vectorssupport active gene and/or small RNA delivery over a period of about 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, 12 months, or longer. Anycombination of these time periods can also be used in the methods of theinvention, e.g., 1 month and 1 week, or 3 months and 2 weeks.

However, in some embodiments, the constructs comprise integratingelements that depend on a retroviral integrase gene, such that theconstruct becomes integrated into the subject's chromosome.Retrotransposition and transposition are additional examples ofmechanisms whereby mobile genetic elements become integrated or insertedinto the chromosome. Plasmids may become integrated into the chromosomeby recombination, and gene editing technologies including CRISPR andTALEN utilize guide RNA sequences and alter chromosomal loci by geneconversion mechanisms.

Constructs may comprise specific promoters for expressing cytokinesinvolved in the maintenance of GD T cells (i.e. IL-2, IL-7, IL-17, andIL-15). For example, promoters that may be incorporated into thedisclosed constructs include but are not limited to TATA-box promoters,CpG-box promoters, CCAAT-box promoters, TTGACA-box promoters, BRE-boxpromoters, INR-box promoters, AT-based promoters, CG-based promoters,ATCG-compact promoters, ATCG-balanced promoters, ATCG-middle promoters,ATCG-less promoters, AT-less promoters, CG-less promoters, AT-spikepromoters, and CG-spike promoters. See Gagniuc and Ionescu-Tirgoviste,Eukaryotic genomes may exhibit up to 10 generic classes of genepromoters, BMC GENOMICS 13:512 (2012).

Therapeutic Vectors

The construct can be delivered via known transfection and/ortransduction vectors, including but not limited to lentiviral vectors,adeno-associated virus, poxvirus, herpesvirus vectors, protein and/orlipid complexes, liposomes, micelles, and the like.

Viral vectors can be preferentially targeted to cell types that areuseful for the disclosed methods (i.e., tumor cells or myeloid cells).Viral vectors can be used to transduce genes into target cells owing tospecific virus envelope-host cell receptor interactions and viralmechanisms for gene expression. As a result, viral vectors have beenused as vehicles for the transfer of genes into many different celltypes including whole embryos, fertilized eggs, isolated tissue samples,tissue targets in situ, and cultured cell lines. The ability tointroduce and express foreign genes in a cell is useful for the study ofgene expression, and the elucidation of cell lineages as well asproviding the potential for therapeutic interventions such as genetherapy, somatic cell reprogramming of induced pluripotent stem cells,and various types of immunotherapy. Viral components from viruses likePapovaviridae (e.g. bovine papillomavirus or BPV) or Herpesviridae (e.g.Epstein Barr Virus or EBV) or Hepadnaviridae (e.g. Hepatitis B Virus orHBV) or pox vectors including vaccinia may be used in the disclosedvectors.

Lentiviral vectors are a preferred type of vector for the disclosedcompositions and methods, although the disclosure is not specificallylimited to lentiviral vectors. Lentivirus is a genus of viruses that candeliver a significant amount of viral nucleic acid into a host cell.Lentiviruses are characterized as having a unique ability toinfect/transduce non-dividing cells, and following transduction,lentiviruses integrate their nucleic acid into the host cell'schromosomes.

Infectious lentiviruses have three main genes coding for the virulenceproteins gag, pol, and env, and two regulatory genes including tat andrev. Depending on the specific serotype and virus, there may beadditional accessory genes that code for proteins involved inregulation, synthesis, and/or processing viral nucleic acids and otherreplicative functions.

Moreover, lentiviruses contain long terminal repeat (LTR) regions, whichmay be approximately 600 nt long. LTRs may be segmented into U3, R, andU5 regions. LTRs can mediate integration of retroviral DNA into the hostchromosome via the action of integrase. Alternatively, withoutfunctioning integrase, the LTRs may be used to circularize the viralnucleic acid.

Viral proteins involved in early stages of lentivirus replicationinclude reverse transcriptase and integrase. Reverse transcriptase isthe virally encoded, RNA-dependent DNA polymerase. The enzyme uses aviral RNA genome as a template for the synthesis of a complementary DNAcopy. Reverse transcriptase also has RNaseH activity for destruction ofthe RNA-template. Integrase binds both the viral cDNA generated byreverse transcriptase and the host DNA. Integrase processes the LTRbefore inserting the viral genome into the host DNA. Tat acts as atrans-activator during transcription to enhance initiation andelongation. The rev responsive element acts post-transcriptionally,regulating mRNA splicing and transport to the cytoplasm.

Viral vectors, in general, comprise glycoproteins and the variousglycoproteins may provide specific affinities. For instance, VSVGpeptides can increase transfection into myeloid cells. Alternatively,viral vectors can also have targeting moieties, such as antibodies,attached to their shell peptides. Targeting antibodies can be specificfor antigens that are overexpressed on a tumor, for instance, likeHER-2, PSA, CEA, M2-PK, and CA19-9.

Other viral vector specificities are also known in the art and can beused to target particular populations of cells. For example, poxvirusvectors target to macrophages and dendritic cells.

Lentiviral Vector System

A lentiviral virion (particle) is expressed by a vector system encodingthe necessary viral proteins to produce a virion (viral particle). Thereis at least one vector containing a nucleic acid sequence encoding thelentiviral pol proteins necessary for reverse transcription andintegration, operably linked to a promoter. In another embodiment, thepol proteins are expressed by multiple vectors. There is also a vectorcontaining a nucleic acid sequence encoding the lentiviral gag proteinsnecessary for forming a viral capsid operably linked to a promoter. Inan embodiment, this gag nucleic acid sequence is on a separate vectorthan at least some of the pol nucleic acid sequence. In anotherembodiment, the gag nucleic acid is on a separate vector from all thepol nucleic acid sequences that encode pol proteins.

Numerous modifications can be made to the vectors, which are used tocreate the particles to further minimize the chance of obtaining wildtype revertants. These include, but are not limited to deletions of theU3 region of the LTR, tat deletions and matrix (MA) deletions.

The gag, pol and env vector(s) do not contain nucleotides from thelentiviral genome that package lentiviral RNA, referred to as thelentiviral packaging sequence.

The vector(s) forming the particle preferably do not contain a nucleicacid sequence from the lentiviral genome that expresses an envelopeprotein. Preferably, a separate vector that contains a nucleic acidsequence encoding an envelope protein operably linked to a promoter isused. This env vector also does not contain a lentiviral packagingsequence. In one embodiment the env nucleic acid sequence encodes alentiviral envelope protein.

In another embodiment the envelope protein is not from the lentivirus,but from a different virus. The resultant particle is referred to as apseudotyped particle. By appropriate selection of envelopes one can“infect” virtually any cell. For example, one can use an env gene thatencodes an envelope protein that targets an endocytic compartment suchas that of the influenza virus, VSV-G, alpha viruses (Semliki forestvirus, Sindbis virus), arenaviruses (lymphocytic choriomeningitisvirus), flaviviruses (tick-borne encephalitis virus, Dengue virus,hepatitis C virus, GB virus), rhabdoviruses (vesicular stomatitis virus,rabies virus), paramyxoviruses (mumps or measles) and orthomyxoviruses(influenza virus). Other envelopes that can preferably be used includethose from Moloney Leukemia Virus such as MLV-E, MLV-A and GALV. Theselatter envelopes are particularly preferred where the host cell is aprimary cell. Other envelope proteins can be selected depending upon thedesired host cell. For example, targeting specific receptors such as adopamine receptor can be used for brain delivery. Another target can bevascular endothelium. These cells can be targeted using a filovirusenvelope. For example, the GP of Ebola, which by post-transcriptionalmodification become the GP, and GP₂ glycoproteins. In anotherembodiment, one can use different lentiviral capsids with a pseudotypedenvelope (for example, FIV or SHIV [U.S. Pat. No. 5,654,195]). A SHIVpseudotyped vector can readily be used in animal models such as monkeys.

As detailed herein, a lentiviral vector system typically includes atleast one helper plasmid comprising at least one of a gag, pol, or revgene. Each of the gag, pol and rev genes may be provided on individualplasmids, or one or more genes may be provided together on the sameplasmid. In one embodiment, the gag, pol, and rev genes are provided onthe same plasmid (e.g., FIG. 2). In another embodiment, the gag and polgenes are provided on a first plasmid and the rev gene is provided on asecond plasmid (e.g., FIG. 3). Accordingly, both 3-vector and 4-vectorsystems can be used to produce a lentivirus as described in the Examplessection and elsewhere herein. The therapeutic vector, the envelopeplasmid and at least one helper plasmid are transfected into a packagingcell line. A non-limiting example of a packaging cell line is the293T/17 HEK cell line. When the therapeutic vector, the envelopeplasmid, and at least one helper plasmid are transfected into thepackaging cell line, a lentiviral particle is ultimately produced.

In another aspect, a lentiviral vector system for expressing alentiviral particle is disclosed. The system includes a lentiviralvector as described herein; an envelope plasmid for expressing anenvelope protein optimized for infecting a cell; and at least one helperplasmid for expressing gag, pol, and rev genes, wherein when thelentiviral vector, the envelope plasmid, and the at least one helperplasmid are transfected into a packaging cell line, a lentiviralparticle is produced by the packaging cell line, wherein the lentiviralparticle is capable of inhibiting production of chemokine receptor CCR5or targeting an HIV RNA sequence.

In another aspect, and as detailed in FIG. 2, the lentiviral vector,which is also referred to herein as a therapeutic vector, can includethe following elements: hybrid 5′ long terminal repeat (RSV/5′ LTR) (SEQID NOS: 11-12), Psi sequence (RNA packaging site) (SEQ ID NO: 13), RRE(Rev-response element) (SEQ ID NO: 14), cPPT (polypurine tract) (SEQ IDNO: 15), H1 promoter (SEQ ID NO: 16), FDPS shRNA (SEQ ID NOS: 1, 2, 3,4), Woodchuck Post-Transcriptional Regulatory Element (WPRE) (SEQ ID NO:17), and 3′ Delta LTR (SEQ ID NO: 18). In another aspect, sequencevariation, by way of substitution, deletion, addition, or mutation canbe used to modify the sequences references herein.

In another aspect, and as detailed herein, a helper plasmid has beendesigned to include the following elements: CAG promoter (SEQ ID NO:19); HIV component gag (SEQ ID NO: 20); HIV component pol (SEQ ID NO:21); HIV Int (SEQ ID NO: 22); HIV RRE (SEQ ID NO: 23); and HIV Rev (SEQID NO: 24). In another aspect, the helper plasmid may be modified toinclude a first helper plasmid for expressing the gag and pol genes, anda second and separate plasmid for expressing the rev gene. In anotheraspect, sequence variation, by way of substitution, deletion, addition,or mutation can be used to modify the sequences references herein.

In another aspect, and as detailed herein, an envelope plasmid has beendesigned to include the following elements being from left to right: RNApolymerase II promoter (CMV) (SEQ ID NO: 25) and vesicular stomatitisvirus G glycoprotein (VSV-G) (SEQ ID NO: 26). In another aspect,sequence variation, by way of substitution, deletion, addition, ormutation can be used to modify the sequences references herein.

In another aspect, the plasmids used for lentiviral packaging can bemodified with similar elements and the intron sequences couldpotentially be removed without loss of vector function. For example, thefollowing elements can replace similar elements in the plasmids thatcomprise the packaging system: Elongation Factor-1 (EF-1),phosphoglycerate kinase (PGK), and ubiquitin C (UbC) promoters canreplace the CMV or CAG promoter. SV40 poly A and bGH poly A can replacethe rabbit beta globin poly A. The HIV sequences in the helper plasmidcan be constructed from different HIV strains or clades. The VSV-Gglycoprotein can be substituted with membrane glycoproteins from felineendogenous virus (RD114), gibbon ape leukemia virus (GALV), Rabies(FUG), lymphocytic choriomeningitis virus (LCMV), influenza A fowlplague virus (FPV), Ross River alphavirus (RRV), murine leukemia virus10A1 (MLV), or Ebola virus (EboV).

Of note, lentiviral packaging systems can be acquired commercially(e.g., Lenti-vpak packaging kit from OriGene Technologies, Inc.,Rockville, Md.), and can also be designed as described herein. Moreover,it is within the skill of a person skilled in the art to substitute ormodify aspects of a lentiviral packaging system to improve any number ofrelevant factors, including the production efficiency of a lentiviralparticle.

Doses and Dosage Forms

The disclosed vectors allow for short, medium, or long-term expressionof genes or sequences of interest and episomal maintenance of thedisclosed vectors. Accordingly, dosing regimens may vary based upon thecondition being treated and the method of administration.

In one embodiment, transduction vectors may be administered to a subjectin need in varying doses. Specifically, a subject may be administeredabout ≥10⁶ infectious doses (where 1 dose is needed on average totransduce 1 target cell). More specifically, a subject may beadministered about ≥10⁷, about ≥10⁸, about ≥10⁹, or about ≥10¹⁰infectious doses, or any number of doses in-between these values. Upperlimits of transduction vector dosing will be determined for each diseaseindication and will depend on toxicity/safety profiles for eachindividual product or product lot.

Additionally, a vector of the present disclosure may be administeredperiodically, such as once or twice a day, or any other suitable timeperiod. For example, vectors may be administered to a subject in needonce a week, once every other week, once every three weeks, once amonth, every other month, every three months, every six months, everynine months, once a year, every eighteen months, every two years, everythirty months, or every three years.

In one embodiment, the disclosed vectors are administered as apharmaceutical composition. In some embodiments, the pharmaceuticalcomposition comprising the disclosed vectors can be formulated in a widevariety of dosage forms, including but not limited to nasal, pulmonary,oral, topical, or parenteral dosage forms for clinical application. Eachof the dosage forms can comprise various solubilizing agents,disintegrating agents, surfactants, fillers, thickeners, binders,diluents such as wetting agents or other pharmaceutically acceptableexcipients. The pharmaceutical composition comprising a vector can alsobe formulated for injection, insufflation, infusion, or intradermalexposure. For instance, an injectable formulation may comprise thedisclosed vectors in an aqueous or non-aqueous solution at a suitable pHand tonicity.

The disclosed vectors may be administered to a subject via directinjection into a tumor site or at a site of infection. In someembodiments, the vectors can be administered systemically. In someembodiments, the vectors can be administered via guided cannulation totissues immediately surrounding the sites of tumor or infection.

The disclosed vector compositions can be administered using anypharmaceutically acceptable method, such as intranasal, buccal,sublingual, oral, rectal, ocular, parenteral (intravenously,intradermally, intramuscularly, subcutaneously, intraperitoneally),pulmonary, intravaginal, locally administered, topically administered,topically administered after scarification, mucosally administered, viaan aerosol, in semi-solid media such as agarose or gelatin, or via abuccal or nasal spray formulation.

Further, the disclosed vector compositions can be formulated into anypharmaceutically acceptable dosage form, such as a solid dosage form,tablet, pill, lozenge, capsule, liquid dispersion, gel, aerosol,pulmonary aerosol, nasal aerosol, ointment, cream, semi-solid dosageform, a solution, an emulsion, and a suspension. Further, thecomposition may be a controlled release formulation, sustained releaseformulation, immediate release formulation, or any combination thereof.Further, the composition may be a transdermal delivery system.

In some embodiments, the pharmaceutical composition comprising a vectorcan be formulated in a solid dosage form for oral administration, andthe solid dosage form can be powders, granules, capsules, tablets orpills. In some embodiments, the solid dosage form can include one ormore excipients such as calcium carbonate, starch, sucrose, lactose,microcrystalline cellulose or gelatin. In addition, the solid dosageform can include, in addition to the excipients, a lubricant such astalc or magnesium stearate. In some embodiments, the oral dosage formcan be immediate release, or a modified release form. Modified releasedosage forms include controlled or extended release, enteric release,and the like. The excipients used in the modified release dosage formsare commonly known to a person of ordinary skill in the art.

In a further embodiment, the pharmaceutical composition comprising avector can be formulated as a sublingual or buccal dosage form. Suchdosage forms comprise sublingual tablets or solution compositions thatare administered under the tongue and buccal tablets that are placedbetween the cheek and gum.

In some embodiments, the pharmaceutical composition comprising a vectorcan be formulated as a nasal dosage form. Such dosage forms of thepresent invention comprise solution, suspension, and gel compositionsfor nasal delivery.

In some embodiments, the pharmaceutical composition comprising a vectorcan be formulated in a liquid dosage form for oral administration, suchas suspensions, emulsions or syrups. In some embodiments, the liquiddosage form can include, in addition to commonly used simple diluentssuch as water and liquid paraffin, various excipients such ashumectants, sweeteners, aromatics or preservatives. In particularembodiments, the composition comprising vectors can be formulated to besuitable for administration to a pediatric patient.

In some embodiment, the pharmaceutical composition can be formulated ina dosage form for parenteral administration, such as sterile aqueoussolutions, suspensions, emulsions, non-aqueous solutions orsuppositories. In some embodiments, the solutions or suspensions caninclude propyleneglycol, polyethyleneglycol, vegetable oils such asolive oil or injectable esters such as ethyl oleate.

The dosage of the pharmaceutical composition can vary depending on thepatient's weight, age, gender, administration time and mode, excretionrate, and the severity of disease.

In some embodiments, the treatment of cancer is accomplished by guideddirect injection of the disclosed vector constructs into tumors, usingneedle, or intravascular cannulation. In some embodiments, the disclosedvectors are administered into the cerebrospinal fluid, blood orlymphatic circulation by venous or arterial cannulation or injection,intradermal delivery, intramuscular delivery or injection into adraining organ near the site of disease.

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples. Allprinted publications referenced herein are specifically incorporated byreference.

Examples Example 1: Development of a Lentiviral Vector System

A lentiviral vector system was developed as summarized in FIG. 4(circularized form). Lentiviral particles were produced in 293T/17 HEKcells (purchased from American Type Culture Collection, Manassas, Va.)following transfection with the therapeutic vector, the envelopeplasmid, and the helper plasmid. The transfection of 293T/17 HEK cells,which produced functional viral particles, employed the reagentPoly(ethylenimine) (PEI) to increase the efficiency of plasmid DNAuptake. The plasmids and DNA were initially added separately in culturemedium without serum in a ratio of 3:1 (mass ratio of PEI to DNA). After2-3 days, cell medium was collected and lentiviral particles werepurified by high-speed centrifugation and/or filtration followed byanion-exchange chromatography. The concentration of lentiviral particlescan be expressed in terms of transducing units/ml (TU/ml). Thedetermination of TU was accomplished by measuring HIV p24 levels inculture fluids (p24 protein is incorporated into lentiviral particles),measuring the number of viral DNA copies per cell by quantitative PCR,or by infecting cells and using light (if the vectors encode luciferaseor fluorescent protein markers).

As mentioned above, a 3-vector system (i.e., a 2-vector lentiviralpackaging system) was designed for the production of lentiviralparticles. A schematic of the 3-vector system is shown in FIG. 2.Briefly, and with reference to FIG. 2, the top-most vector is a helperplasmid, which, in this case, includes Rev. The vector appearing in themiddle of FIG. 2 is the envelope plasmid. The bottom-most vector is thetherapeutic vector, as described herein.

Referring more specifically to FIG. 2, the Helper plus Rev plasmidincludes a CAG enhancer (SEQ ID NO: 27); a CAG promoter (SEQ ID NO: 19);a chicken beta actin intron (SEQ ID NO: 28); a HIV gag (SEQ ID NO: 20);a HIV Pol (SEQ ID NO: 21); a HIV Int (SEQ ID NO: 22); a HIV RRE (SEQ IDNO: 23); a HIV Rev (SEQ ID NO: 24); and a rabbit beta globin poly A (SEQID NO: 29).

The Envelope plasmid includes a CMV promoter (SEQ ID NO: 25); a betaglobin intron (SEQ ID NO: 30); a VSV-G (SEQ ID NO: 28); and a rabbitbeta globin poly A (SEQ ID NO: 31).

Synthesis of a 2-Vector Lentiviral Packaging System Including Helper(Plus Rev) and Envelope Plasmids.

Materials and Methods:

Construction of the Helper Plasmid:

The helper plasmid was constructed by initial PCR amplification of a DNAfragment from the pNL4-3 HIV plasmid (NIH Aids Reagent Program)containing Gag, Pol, and Integrase genes. Primers were designed toamplify the fragment with EcoRI and NotI restriction sites which couldbe used to insert at the same sites in the pCDNA3 plasmid (Invitrogen).The forward primer was (5′-TAAGCAGAATTCATGAATTTGCCAGGAAGAT-3′) (SEQ IDNO: 32) and reverse primer was(5′-CCATACAATGAATGGACACTAGGCGGCCGCACGAAT-3′) (SEQ ID NO: 33).

The sequence for the Gag, Pol, Integrase fragment was as follows:

(SEQ ID NO: 34) GAATTCATGAATTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAAGACAGTATGATCAGATACTCATAGAAATCTGCGGACATAAAGCTATAGGTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGCTGCACTTTAAATTTTCCCATTAGTCCTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGAAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGGGAAGTTCAATTAGGAATACCACATCCTGCAGGGTTAAAACAGAAAAAATCAGTAACAGTACTGGATGTGGGCGATGCATATTTTTCAGTTCCCTTAGATAAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCCAGTGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTCATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAACTGAGACAACATCTGTTGAGGTGGGGATTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAGGACAGCTGGACTGTCAATGACATACAGAAATTAGTGGGAAAATTGAATTGGGCAAGTCAGATTTATGCAGGGATTAAAGTAAGGCAATTATGTAAACTTCTTAGGGGAACCAAAGCACTAACAGAAGTAGTACCACTAACAGAAGAAGCAGAGCTAGAACTGGCAGAAAACAGGGAGATTCTAAAAGAACCGGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTGAAAACAGGAAAGTATGCAAGAATGAAGGGTGCCCACACTAATGATGTGAAACAATTAACAGAGGCAGTACAAAAAATAGCCACAGAAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAATTACCCATACAAAAGGAAACATGGGAAGCATGGTGGACAGAGTATTGGCAAGCCACCTGGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCTTAGTGAAGTTATGGTACCAGTTAGAGAAAGAACCCATAATAGGAGCAGAAACTTTCTATGTAGATGGGGCAGCCAATAGGGAAACTAAATTAGGAAAAGCAGGATATGTAACTGACAGAGGAAGACAAAAAGTTGTCCCCCTAACGGACACAACAAATCAGAAGACTGAGTTACAAGCAATTCATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTGACAGACTCACAATATGCATTGGGAATCATTCAAGCACAACCAGATAAGAGTGAATCAGAGTTAGTCAGTCAAATAATAGAGCAGTTAATAAAAAAGGAAAAAGTCTACCTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAAGTAGATAAATGGGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAGATGGAATAGATAAGGCCCAAGAAGAACATGAGAAATATCACAGTAATTGGAGAGCAATGGCTAGGGATTTTAACCTACCACCTGTAGTAGCAAAAGAAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGGGAAGCCATGCATGGACAAGTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGAGGGCCAGTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATCAGGGATTATGGAAAACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGGATTAA

Next, a DNA fragment containing the Rev, RRE, and rabbit beta globinpoly A sequence with XbaI and XmaI flanking restriction sites wassynthesized by MWG Operon. The DNA fragment was then inserted into theplasmid at the XbaI and XmaI restriction sites The DNA sequence was asfollows:

(SEQ ID NO: 35) TCTAGAATGGCAGGAAGAAGCGGAGACAGCGACGAAGAGCTCATCAGAACAGTCAGACTCATCAAGCTTCTCTATCAAAGCAACCCACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTGGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAATAGAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCATATGCTGGCTGCCATGAACAAAGGTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGAAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCAGCGGCCGCCCCGGG

Finally, the CMV promoter of pCDNA3.1 was replaced with the CAGenhancer/promoter plus a chicken beta actin intron sequence. A DNAfragment containing the CAG enhancer/promoter/intron sequence with MluIand EcoRI flanking restriction sites was synthesized by MWG Operon. TheDNA fragment was then inserted into the plasmid at the MluI and EcoRIrestriction sites. The DNA sequence was as follows:

(SEQ ID NO: 36) ACGCGTTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCATCTCCAGCCTCGGGGCTGCCGCAGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTG ACCGGCGGGAATTC

Construction of the VSV-G Envelope Plasmid:

The vesicular stomatitis Indiana virus glycoprotein (VSV-G) sequence wassynthesized by MWG Operon with flanking EcoRI restriction sites. The DNAfragment was then inserted into the pCDNA3.1 plasmid (Invitrogen) at theEcoRI restriction site and the correct orientation was determined bysequencing using a CMV specific primer. The DNA sequence was as follows:

(SEQ ID NO: 37) GAATTCATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGCAAGTTCACCATAGTTTTTCCACACAACCAAAAAGGAAACTGGAAAAATGTTCCTTCTAATTACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGCATAATGACTTAATAGGCACAGCCTTACAAGTCAAAATGCCCAAGAGTCACAAGGCTATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCCATCCGATCCTTCACTCCATCTGTAGAACAATGCAAGGAAAGCATTGAACAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGTTGTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTGACTCCTCACCATGTGCTGGTTGATGAATACACAGGAGAATGGGTTGATTCACAGTTCATCAACGGAAAATGCAGCAATTACATATGCCCCACTGTCCATAACTCTACAACCTGGCATTCTGACTATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCATGGACATCACCTTCTTCTCAGAGGACGGAGAGCTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAGAAGTAACTACTTTGCTTATGAAACTGGAGGCAAGGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCTCAGACCTCAGTGGATGTAAGTCTAATTCAGGACGTTGAGAGGATCTTGGATTATTCCCTCTGCCAAGAAACCTGGAGCAAAATCAGAGCGGGTCTTCCAATCTCTCCAGTGGATCTCAGCTATCTTGCTCCTAAAAACCCAGGAACCGGTCCTGCTTTCACCATAATCAATGGTACCCTAAAATACTTTGAGACCAGATACATCAGAGTCGATATTGCTGCTCCAATCCTCTCAAGAATGGTCGGAATGATCAGTGGAACTACCACAGAAAGGGAACTGTGGGATGACTGGGCACCATATGAAGACGTGGAAATTGGACCCAATGGAGTTCTGAGGACCAGTTCAGGATATAAGTTTCCTTTATACATGATTGGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTCAAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTATTTTTTGGTGATACTGGGCTATCCAAAAATCCAATCGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTTCTCCGAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAAAGACAGATTTATACAGACATAGAGATGAGAATTC

A 4-vector system (i.e., a 3-vector lentiviral packaging system) hasalso been designed and produced using the methods and materialsdescribed herein. A schematic of the 4-vector system is shown in FIG. 3.Briefly, and with reference to FIG. 3, the top-most vector is a helperplasmid, which, in this case, does not include Rev. The vector secondfrom the top is a separate Rev plasmid. The vector second from thebottom is the envelope plasmid. The bottom-most vector is the previouslydescribed therapeutic vector.

Referring, in part, to FIG. 2, the Helper plasmid includes a CAGenhancer (SEQ ID NO: 27); a CAG promoter (SEQ ID NO: 19); a chicken betaactin intron (SEQ ID NO: 28); a HIV gag (SEQ ID NO: 20); a HIV Pol (SEQID NO: 21); a HIV Int (SEQ ID NO: 22); a HIV RRE (SEQ ID NO: 23); and arabbit beta globin poly A (SEQ ID NO: 29).

The Rev plasmid includes a RSV promoter (SEQ ID NO: 38); a HIV Rev (SEQID NO: 39); and a rabbit beta globin poly A (SEQ ID NO: 29).

The Envelope plasmid includes a CMV promoter (SEQ ID NO: 25); a betaglobin intron (SEQ ID NO: 30); a VSV-G (SEQ ID NO: 28); and a rabbitbeta globin poly A (SEQ ID NO: 29).

Synthesis of a 3-Vector Lentiviral Packaging System Including Helper,Rev, and Envelope Plasmids.

Materials and Methods:

Construction of the Helper Plasmid without Rev:

The Helper plasmid without Rev was constructed by inserting a DNAfragment containing the RRE and rabbit beta globin poly A sequence. Thissequence was synthesized by MWG Operon with flanking XbaI and XmaIrestriction sites. The RRE/rabbit poly A beta globin sequence was theninserted into the Helper plasmid at the XbaI and XmaI restriction sites.The DNA sequence is as follows:

(SEQ ID NO: 67) TCTAGAAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCATATGCTGGCTGCCATGAACAAAGGTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGAAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCACCCGGG 

Construction of the Rev Plasmid:

The RSV promoter and HIV Rev sequence was synthesized as a single DNAfragment by MWG Operon with flanking MfeI and XbaI restriction sites.The DNA fragment was then inserted into the pCDNA3.1 plasmid(Invitrogen) at the MfeI and XbaI restriction sites in which the CMVpromoter is replaced with the RSV promoter. The DNA sequence was asfollows:

(SEQ ID NO: 40) CAATTGCGATGTACGGGCCAGATATACGCGTATCTGAGGGGACTAGGGTGTGTTTAGGCGAAAAGCGGGGCTTCGGTTGTACGCGGTTAGGAGTCCCCTCAGGATATAGTAGTTTCGCTTTTGCATAGGGAGGGGGAAATGTAGTCTTATGCAATACACTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACAGGTCTGACATGGATTGGACGAACCACTGAATTCCGCATTGCAGAGATAATTGTATTTAAGTGCCTAGCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTCGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAAGCTAGCGATTAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGAAGAACTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACCCACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTAGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAATAGTCTAGA

The plasmids for the 2-vector and 3-vector packaging systems could bemodified with similar elements and the intron sequences couldpotentially be removed without loss of vector function. For example, thefollowing elements could replace similar elements in the 2-vector and3-vector packaging system:

Promoters: Elongation Factor-1 (EF-1) (SEQ ID NO: 41), phosphoglyceratekinase (PGK) (SEQ ID NO: 42), and ubiquitin C (UbC) (SEQ ID NO: 43) canreplace the CMV (SEQ ID NO: 25) or CAG promoter (SEQ ID NO: 19). Thesesequences can also be further varied by addition, substitution, deletionor mutation.

Poly A sequences: SV40 poly A (SEQ ID NO: 44) and bGH poly A (SEQ ID NO:45) can replace the rabbit beta globin poly A (SEQ ID NO: 29). Thesesequences can also be further varied by addition, substitution, deletionor mutation.

HIV Gag, Pol, and Integrase sequences: The HIV sequences in the Helperplasmid can be constructed from different HIV strains or clades. Forexample, HIV Gag (SEQ ID NO: 20); HIV Pol (SEQ ID NO: 21); and HIV Int(SEQ ID NO: 22) from the Bal strain can be interchanged with the gag,pol, and int sequences contained in the helper/helper plus Rev plasmidsas outlined herein. These sequences can also be further varied byaddition, substitution, deletion or mutation.

Envelope: The VSV-G glycoprotein can be substituted with membraneglycoproteins from feline endogenous virus (RD114) (SEQ ID NO: 46),gibbon ape leukemia virus (GALV) (SEQ ID NO: 47), Rabies (FUG) (SEQ IDNO: 48), lymphocytic choriomeningitis virus (LCMV) (SEQ ID NO: 49),influenza A fowl plague virus (FPV) (SEQ ID NO: 50), Ross Riveralphavirus (RRV) (SEQ ID NO: 51), murine leukemia virus 10A1 (MLV) (SEQID NO: 52), or Ebola virus (EboV) (SEQ ID NO: 53). Sequences for theseenvelopes are identified in the sequence portion herein. Further, thesesequences can also be further varied by addition, substitution, deletionor mutation.

In summary, the 3-vector versus 4-vector systems can be compared andcontrasted, in part, as follows. The 3-vector lentiviral vector systemcontains: 1. Helper plasmid: HIV Gag, Pol, Integrase, and Rev/Tat; 2.Envelope plasmid: VSV-G/FUG envelope; and 3. Therapeutic vector: RSV5′LTR, Psi Packaging Signal, Gag fragment, RRE, Env fragment, cPPT,WPRE, and 3′6 LTR. The 4-vector lentiviral vector system contains: 1.Helper plasmid: HIV Gag, Pol, and Integrase; 2. Rev plasmid: Rev; 3.Envelope plasmid: VSV-G/FUG envelope; and 4. Therapeutic vector: RSV5′LTR, Psi Packaging Signal, Gag fragment, RRE, Env fragment, cPPT,WPRE, and 3′ delta LTR. Sequences corresponding with the above elementsare identified in the sequence listings portion herein.

Example 2: Development of a Lentiviral Vector that Expresses FDPS

The purpose of this Example was to develop an FDPS lentivirus vector.

Inhibitory RNA Design:

The sequence of Homo sapiens Famesyl diphosphate synthase (FDPS)(NM_002004.3) mRNA was used to search for potential siRNA or shRNAcandidates to knockdown FDPS levels in human cells. Potential RNAinterference sequences were chosen from candidates selected by siRNA orshRNA design programs such as from GPP Web Portal hosted by the BroadInstitute (http://portals.broadinstitute.org/gpp/public/) or theBLOCK-iT RNAi Designer from Thermo Scientific(https://maidesigner.thermofisher.com/maiexpress/). Individual selectedshRNA sequences were inserted into a lentiviral vector immediately 3prime to a RNA polymerase III promoter such as H1 (SEQ ID NO: 16), U6(SEQ ID NO: 54), or 7SK (SEQ ID NO: 55) to regulate shRNA expression.These lentivirus shRNA constructs were used to transduce cells andmeasure the change in specific mRNA levels. The shRNA most potent forreducing mRNA levels were embedded individually within a microRNAbackbone to allow for expression by either the EF-1alpha or CMV RNApolymerase II promoters. The microRNA backbone was selected frommirbase.org. RNA sequences were also synthesized as synthetic siRNAoligonucleotides and introduced directly into cells without using alentiviral vector.

Vector Construction:

For FDPS shRNA, oligonucleotide sequences containing BamHI and EcoRIrestriction sites were synthesized by Eurofins MWG Operon. Overlappingsense and antisense oligonucleotide sequences were mixed and annealedduring cooling from 70 degrees Celsius to room temperature. Thelentiviral vector was digested with the restriction enzymes BamHI andEcoRI for one hour at 37 degrees Celsius. The digested lentiviral vectorwas purified by agarose gel electrophoresis and extracted from the gelusing a DNA gel extraction kit from Thermo Scientific. The DNAconcentrations were determined and vector to oligo (3:1 ratio) weremixed, allowed to anneal, and ligated. The ligation reaction wasperformed with T4 DNA ligase for 30 minutes at room temperature. 2.5microliters of the ligation mix were added to 25 microliters of STBL3competent bacterial cells. Transformation was achieved after heat-shockat 42 degrees Celsius. Bacterial cells were spread on agar platescontaining ampicillin and drug-resistant colonies (indicating thepresence of ampicillin-resistance plasmids) were recovered and expandedin LB broth. To check for insertion of the oligo sequences, plasmid DNAwas extracted from harvested bacteria cultures with the ThermoScientific DNA mini prep kit. Insertion of shRNA sequences in thelentiviral vector was verified by DNA sequencing using a specific primerfor the promoter used to regulate shRNA expression. Using the followingtarget sequences, exemplary shRNA sequences were determined toknock-down FDPS:

GTCCTGGAGTACAATGCCATT (FDPS target sequence #1; SEQ ID NO: 56);GTCCTGGAGTACAATGCCATTCTCGAGAATGGCATTGTACTCCAGGACTTTTT (FDPS shRNA sequence #1; SEQ ID NO: 1);GCAGGATTTCGTTCAGCACTT (FDPS target sequence #2; SEQ ID NO: 57);GCAGGATTTCGTTCAGCACTTCTCGAGAAGTGCTGAACGAAATCCTGCTTTTT (FDPS shRNA sequence #2; SEQ ID NO: 2);GCCATGTACATGGCAGGAATT (FDPS target sequence #3; SEQ ID NO: 58);GCCATGTACATGGCAGGAATTCTCGAGAATTCCTGCCATGTACATGGCTTTTT (FDPS shRNA sequence #3; SEQ ID NO: 3);GCAGAAGGAGGCTGAGAAAGT (FDPS target sequence #4; SEQ ID NO: 59); andGCAGAAGGAGGCTGAGAAAGTCTCGAGACTTTCTCAGCCTCCTTCTGCTTTTT (FDPS shRNA sequence #4; SEQ ID NO: 4).

shRNA sequences were then assembled into a synthetic microRNA (miR)under control of the EF-1 alpha promoter. Briefly, a miR hairpinsequences, such as miR30, miR21, or miR185 as detailed below, wasobtained from mirbase.org. The 19-22mer shRNA target sequence was usedto construct the synthetic miR sequence. The miR sequence was arrangedas an anti-sense-target-sequence-hairpin loop sequence (specific foreach microRNA)-sense target sequence.

The following miR sequences were developed:

AAGGTATATTGCTGTTGACAGTGAGCGACACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGAGGCTGAGAAAGTGCTGCCTACTGCCTCGGACTTCAAGGGGCT (miR30 FDPS sequence #1; SEQ ID NO: 5)AAGGTATATTGCTGTTGACAGTGAGCGACACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGGCTGAGAAAGTGCTGCCTACTGCCTCGGACTTCAAGGGGCT (miR30 FDPS sequence #2; SEQ ID NO: 6)TGCTGTTGACAGTGAGCGACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGAGGCTGAGAAAGTTGCCTACTGCCTCGGA (miR30FDPS sequence #3; SEQ ID NO: 7)CCTGGAGGCTTGCTGAAGGCTGTATGCTGACTTTCTCAGCCTCCTTCTGCTTTTGGCCACTGACTGAGCAGAAGGGCTGAGAAAGTCAGGACACAAGGCCTGTTACTAGCACTCA (miR155 FDPS sequence #1; SEQ ID NO: 8)CATCTCCATGGCTGTACCACCTTGTCGGGACTTTCTCAGCCTCCTTCTGCCTGTTGAATCTCATGGCAGAAGGAGGCGAGAAAGTCTGACATTTTGGTATCTTTCATCTGACCA (miR21 FDPS sequence #1; SEQ ID NO: 9)GGGCCTGGCTCGAGCAGGGGGCGAGGGATACTTTCTCAGCCTCCTTCTGCTGGTCCCCTCCCCGCAGAAGGAGGCTGAGAAAGTCCTTCCCTCCCAATGACCGCGTCTTCGTCG (miR185 FDPS sequence #1; SEQ ID NO: 10)

Example 3—Knock-Down of FDPS for 3 Days in THP1 Monocytic Leukemia byshRNA #4

This Example illustrates that knock-down of FDPS in THP1 monocyticleukemia cells by lentiviral (LV)-expressing FDPS shRNA #4 stimulatesTNF-α expression in gamma delta T cells, as shown in FIG. 5.

THP1 cells (1×10⁵ cells) were transduced with LV-control or LV-FDPSshRNA #4 for 3 days. Two days after transduction, cells were treatedwith or without 1 μM zoledronic acid. After 24 hours, the transducedTHP-1 cells were co-cultured with 5×10⁵ PBMC cells and IL-2 in a roundbottom 96 well plate for 4 hours. The PBMC cells were pre-stimulatedwith zoledronic acid and IL-2 for 11 days to expand Vγ9Vδ2 T cells.After staining for Vγ9Vδ2 and TNF-α using fluorophore-conjugated antiTCR-Vδ2 and anti-TNF-α antibody, cells were analyzed via flow cytometry.Live cells were gated, and Vδ2+ and TNF-α+ cells were selected on a dotblot. The activated cytotoxic Vγ9Vδ2 T cells appeared in the upper rightquadrant of flow cytograms. Without zoledronic acid, LV-controlstimulated 3.1% of TNF-α expressing Vγ9Vδ2 T cells and LV-FDPS shRNA #4stimulated 5%. With zoledronic acid treatment, LV-control stimulated7.2% of TNF-α expressing Vγ9Vδ2 T cells and LV-FDPS shRNA #4 stimulated56.2%.

Example 4—Knock-Down of FDPS for 14 Days in THP1 Leukemia Cells by shRNA#4

This Example illustrates that Knock-down of FDPS for 14 days in THP1leukemia cells by lentiviral (LV)-expressing FDPS shRNA #4 stimulatesTNF-α expression in GD T cells, as shown in FIG. 6.

THP1 cells (1×10⁵ cells) were transduced with LV-control or LV-FDPSshRNA #4 for 14 days. Two days after transduction, cells were treatedwith or without 1 uM zoledronic acid. After 24 hours, the transducedTHP-1 cells were co-cultured with 5×10⁵ PBMC cells and IL-2 in a roundbottom 96 well plate for 4 hours. The PBMC cells were pre-stimulatedwith zoledronic acid and IL-2 for 11 days to expand Vγ9Vδ2 T cells.After staining for Vγ9Vδ2 and TNF-α using fluorophore-conjugated antiTCR-Vδ2 and anti-TNF-α antibody, cells were analyzed via flow cytometry.Live cells were gated, and Vδ2+ and TNF-α+ cells were selected on a dotblot. The activated cytotoxic Vγ9Vδ2 T cells appeared in the upper rightquadrant of flow cytograms. Without zoledronic acid, LV-controlstimulated 0.9% of TNF-α expressing Vγ9Vδ2 T cells and LV-FDPS shRNA #4(SEQ ID NO: 4) stimulated 15.9%. With zoledronic acid treatment,LV-control stimulated 4.7% of TNF-α expressing Vγ9Vδ2 T cells andLV-FDPS shRNA #4 (SEQ ID NO: 4) stimulated 76.2%.

Example 5—Knock-Down of FDPS for 3 Days in PC3 Prostate Carcinoma Cellsby shRNA #1

This Example illustrates that knock-down of FDPS for 3 days in PC3prostate carcinoma cells by lentiviral (LV)-expressing FDPS shRNA #1stimulates TNF-α expression in GD T cells, as shown in FIG. 7.

PC3 cells were transduced with LV-control or LV-FDPS shRNA #1 (SEQ IDNO: 1) for 3 days. Two days after transduction, cells were treated withor without 1 uM zoledronic acid. After 24 hours, the transduced PC3cells were co-cultured with 5×10⁵ PBMC cells and IL-2 in a round bottom96 well plate for 4 hours. The PBMC cells were pre-stimulated withzoledronic acid and IL-2 for 11 days to expand Vγ9Vδ2 T cells. Afterstaining for Vγ9Vδ2 and TNF-α using fluorophore-conjugated anti TCR-Vδ2and anti-TNF-α antibody, cells were analyzed via flow cytometry. Livecells were gated, and Vδ2+ and TNF-α+ cells were selected on a dot blot.The activated cytotoxic Vγ9Vδ2 T cells appeared in the upper rightquadrant of flow cytograms. Without zoledronic acid, LV-controlstimulated 0.2% of TNF-α expressing Vγ9Vδ2 T cells and LV-FDPS shRNA #1stimulated 0.5%. With zoledronic acid treatment, LV-control stimulated1.7% of TNF-α expressing Vγ9Vδ2 T cells and LV-FDPS shRNA #1 (SEQ IDNO: 1) stimulated 32.2%.

Example 6—Knock-Down of FDPS for 3 Days in PC3 Prostate Carcinoma Cellsby shRNA #4

This Example illustrates that Knock-down of FDPS for 3 days in PC3prostate carcinoma cells by lentiviral (LV)-expressing FDPS shRNA #4stimulates TNF-α expression in GD T cells, as shown in FIG. 8.

PC3 cells were transduced with LV-control or LV-FDPS shRNA #4 (SEQ IDNO: 4) for 3 days. Two days after transduction, cells were treated withor without 1 uM zoledronic acid. After 24 hours, the transduced PC3cells were co-cultured with 5×10⁵ PBMC cells and IL-2 in a round bottom96 well plate for 4 hours. The PBMC cells were pre-stimulated withzoledronic acid and IL-2 for 11 days to expand Vγ9Vδ2 T cells. Afterstaining for Vγ9Vδ2 and TNF-α using fluorophore-conjugated anti TCR-Vδ2and anti-TNF-α antibody, cells were analyzed via flow cytometry. Livecells were gated, and Vδ2+ and TNF-α+ cells were selected on a dot blot.The activated cytotoxic Vγ9Vδ2 T cells appeared in the upper rightquadrant of flow cytograms. Without zoledronic acid, LV-controlstimulated 0.5% of TNF-α expressing Vγ9Vδ2 T cells and LV-FDPS shRNA #4(SEQ ID NO: 4) stimulated 1.9%. With zoledronic acid treatment,LV-control stimulated 2.1% of TNF-α expressing Vγ9Vδ2 T cells andLV-FDPS shRNA #4 stimulated 28.7%.

Example 7—Knock-Down of FDPS for 3 Days in HepG2 Liver Carcinoma Cellsby shRNA #1 and #4

This Example illustrates that Knock-down of FDPS for 3 days in HepG2liver carcinoma cells by lentiviral (LV)-expressing FDPS shRNA #1 (SEQID NO: 1) and shRNA#4 (SEQ ID NO: 4) stimulates TNF-α expression in GD Tcells, as shown in FIG. 9. HepG2 cells were transduced with LV-control,LV-FDPS shRNA #1 (SEQ ID NO: 1), or LV-FDPS shRNA #4 (SEQ ID NO: 4) for3 days. Two days after transduction, cells were treated with or without1 uM zoledronic acid. After 24 hours, the transduced HepG2 cells wereco-cultured with 5×10⁵ PBMC cells and IL-2 in a round bottom 96 wellplate for 4 hours. The PBMC cells were pre-stimulated with zoledronicacid and IL-2 for 11 days to expand Vγ9Vδ2 T cells. After staining forVγ9Vδ2 and TNF-α using fluorophore-conjugated anti TCR-Vδ2 andanti-TNF-α antibody, cells were analyzed via flow cytometry. Live cellswere gated, and Vδ2+ and TNF-α+ cells were selected on a dot blot. Theactivated cytotoxic Vγ9Vδ2 T cells appeared in the upper right quadrantof flow cytograms. Without zoledronic acid, LV-control stimulated 0.4%of TNF-α expressing Vγ9Vδ2 T cells and LV-FDPS shRNA #1 (SEQ ID NO: 1)and #4 (SEQ ID NO: 4) stimulated 0.7% and 0.9%, respectively. Withzoledronic acid treatment, LV-control stimulated 6.9% of TNF-αexpressing Vγ9Vδ2 T cells and LV-FDPS shRNA #1 and #4 stimulated 7.6%and 21.1%, respectively.

Example 8—Knock-Down of FDPS for 3 Days in THP1 Leukemia by microRNA-30

This Example illustrates that Knock-down of FDPS for 3 days in THP1leukemia cells by lentiviral (LV)-expressing FDPS-targeted syntheticmicroRNA-30 stimulates TNF-α expression in gamma delta T cells, as shownin FIG. 10.

THP1 cells (1×10⁵ cells) were transduced with LV-control or LV-miR30FDPS #1 (SEQ ID NO: 5) for 3 days. Two days after transduction, cellswere treated with or without 1 uM zoledronic acid. After 24 hours, thetransduced THP-1 cells were co-cultured with 5×10⁵ PBMC cells and IL-2in a round bottom 96 well plate for 4 hours. The PBMC cells werepre-stimulated with zoledronic acid and IL-2 for 11 days to expandVγ9Vδ2 T cells. After staining for Vγ9Vδ2 and TNF-α usingfluorophore-conjugated anti TCR-Vδ2 and anti-TNF-α antibody, cells wereanalyzed via flow cytometry. Live cells were gated, and Vδ2+ and TNF-α+cells were selected on a dot blot. The activated cytotoxic Vγ9Vδ2 Tcells appeared in the upper right quadrant of flow cytograms. Withoutzoledronic acid, LV-control stimulated 0.2% of TNF-α expressing Vγ9Vδ2 Tcells and LV-miR30 FDPS stimulated 8.1%. With zoledronic acid treatment,LV-control stimulated 5.3% of TNF-α expressing Vγ9Vδ2 T cells andLV-miR30 FDPS #1 (SEQ ID NO: 5) stimulated 67.3%.

Example 9: E:T Ratios Resulting from Mixture of THP-1 Cells, CulturedHuman GD T Cells, and/or Zometa (Zol)

This Example demonstrates results from mixing treated THP-1 monocytoidtumor cells with cultured human GD T cells, as shown in FIG. 11.

The monocytoid cell line THP-1 was treated with control lentivirusvector (LV), LV suppressing famesyl diphosphate synthase gene expression(LV-FDPS), zoledronic acid (Zol) or combinations. The legend, as shownin FIG. 11, was: lentiviral control vectors (LV-Control), lentiviralvectors expressing microRNA to down regulate FDPS (LV-FPPS), Zometa(Zol), Zometa plus lentiviral control (Zol+LV-Control), or Zometa pluslentiviral vectors expressing microRNA to down regulate FPPS(Zol+LV-FPPS).

Human GD T cells were cultured from an anonymous donor and added totreated THP-1 cells in 4:1. 2:1 or 1:1 ratios (GD T:THP-1) for 4 hours.Cell killing was measured by a fluorescence assay. When THP-1 cells weretreated with a combination of LV-FDPS and Zol, cytotoxic T cell killingby GD T cells was increased greatly compared to either treatment alone.When LV-FDPS treatment alone was compared to Zol treatment alone, theLV-FDPS lead to greater killing but was >3-fold below tumor cell killingafter combination treatment. The combined LV-FDPS plus Zol treatmentcaused nearly 70% tumor cell killing with 4:1 ratio; this was more than3-fold higher than the second best treatment (LV-FDPS alone).

Example 10—Lentiviral-Delivered shRNA-Based RNA Interference Targetingthe Human Farnesyl Diphosphate Synthase (FDPS) Gene

HepG2 human hepatocellular carcinoma cells were infected with lentiviralvectors containing the H1 promoter and either a non-targeting or fourdifferent FDPS shRNA sequences, as shown in FIG. 12. After 48 hours, RNAwas extracted from the cells and converted to cDNA. Expression of FDPScDNA was determined by quantitative PCR using SYBR Green and FDPSprimers. FDPS expression was normalized to actin levels for each sample.

FDPS-targeting lentiviral vectors containing the H1 promoter and eithera non-targeting sequence(5′-GCCGCTTTGTAGGATAGAGCTCGAGCTCTATCCTACAAAGCGGCTTTTT-3′) (SEQ ID NO:60) or one of four different FDPS shRNA sequencesGTCCTGGAGTACAATGCCATTCTCGAGAATGGCATTGTACTCCAGGACTTTTT (FDPS shRNAsequence #1; SEQ ID NO: 1);GCAGGATTTCGTTCAGCACTTCTCGAGAAGTGCTGAACGAAATCCTGCTTTTT (FDPS shRNAsequence #2; SEQ ID NO: 2);GCCATGTACATGGCAGGAATTCTCGAGAATTCCTGCCATGTACATGGCTTTTT (FDPS shRNAsequence #3; SEQ ID NO: 3); andGCAGAAGGAGGCTGAGAAAGTCTCGAGACTTTCTCAGCCTCCTTCTGCTTTTT (FDPS shRNAsequence #4; SEQ ID NO: 4) were produced in 293 T cells.

HepG2 human hepatocellular carcinoma cells were then infected withlentiviral vectors to determine the efficacy of FDPS knock-down. After48 hours, RNA was extracted from the cells using the RNeasy RNAisolation kit (Qiagen) and converted to cDNA with the SuperScript VILOcDNA synthesis kit (Thermo Scientific). Expression of FDPS cDNA wasdetermined by quantitative PCR on an Applied Biosystems StepOne qPCRmachine using a SYBR Green PCR mix (Thermo Scientific) and FDPS primers(Forward primer: 5′-AGGAATTGATGGCGAGAAGG-3′ (SEQ ID NO: 61) and Reverseprimer: 5′-CCCAAAGAGGTCAAGGTAATCA-3′ (SEQ ID NO: 62)). FDPS expressionwas normalized to actin levels for each sample using the actin primers(Forward primer: 5′-AGCGCGGCTACAGCTTCA-3′ (SEQ ID NO: 63) and Reverseprimer: 5′-GGCGACGTAGCACAGCTTCT-3′) (SEQ ID NO: 64). The relative FDPSRNA expression of the shCon sample is set at 100%. There was an 85%(FDPS sequence #1), 89% (FDPS sequence #2), 46% (FDPS sequence #3), and98% (FDPS sequence #4) decrease in FDPS expression.

Example 11—Lentiviral-Delivered miR-Based RNA Interference Targeting theHuman Farnesyl Diphosphate Synthase (FDPS) Gene

As shown in FIG. 13, HepG2 human hepatocellular carcinoma cells wereinfected with lentiviral vectors containing either the H1 promoter (SEQID NO: 16) the FDPS shRNA #4 (SEQ ID NO: 4) sequence or the EF-1αpromoter (SEQ ID NO: 41) and miR30-based FDPS sequences. After 48 hours,cells were lysed and an immunoblot was performed using an anti-FDPS(Thermo Scientific) and an anti-actin (Sigma) antibody as a proteinloading control.

More specifically, HepG2 human hepatocellular carcinoma cells wereinfected with lentiviral vectors containing either the H1 promoter (SEQID NO: 16) and the FDPS shRNA sequenceGCAGAAGGAGGCTGAGAAAGTCTCGAGACTTTCTCAGCCTCCTTCTGCTTTTT (FDPS shRNAsequence #4; SEQ ID NO: 4) or the EF-1alpha promoter (SEQ ID NO: 41) andmiR30-based FDPS sequencesAAGGTATATTGCTGTTGACAGTGAGCGACACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGAGGCTGAGAAAGTGCTGCCTACTGCCTCGGACTTCA AGGGGCT (miR30FDPS sequence #1; SEQ ID NO: 5) andAAGGTATATTGCTGTTGACAGTGAGCGACACTTTCTCAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAAGGGCTGAGAAAGTGCTGCCTACTGCCTCGGACTTCAAG GGGCT (miR30 FDPSsequence #2; SEQ ID NO: 6).

After 48 hours, cells were lysed with NP-40 lysis buffer and protein wasquantified with the Bio-Rad protein assay reagent. Protein samples at 50micrograms were electrophoresed on 4-12% Bis-Tris gels (ThermoScientific and transferred to PVDF membranes (EMD Millipore). Animmunoblot was performed using an anti-FDPS (Thermo Scientific) and ananti-actin (Sigma) antibody as a protein loading control. Antibodieswere bound with HRP-conjugated secondary antibodies and detected with aLicor c-DiGit Blot scanner using the Immobilon Western ECL reagent (EMDMillipore). The densitometry of the immunoblot bands were quantifiedwith the NIH image software. The LV control with the EF-1 promoter wasset at 100%. There was a 68% (LV-shFDPS #4), 43% (LV-miR FDPS #1), and38% (LV-miR FDPS #3) reduction of FDPS protein expression.

Example 12—Knock-Down of FDPS for 3 Days in HepG2 Liver Carcinoma Cellsby Adeno-Associated Virus (AAV)-Expressing FDPS shRNA #4

This Example illustrates that knock-down of FDPS for 3 days in HepG2liver carcinoma cells by adeno-associated virus (AAV)-expressing FDPSshRNA #4 (SEQ ID NO: 4) stimulates TNF-α expression in GD T cells (FIG.14, Panel B).

HepG2 cells were transduced with control or AAV-FDPS shRNA #4 (SEQ IDNO: 8) for 3 days. Two days after transduction, cells were treated withor without 1 uM zoledronic acid. After 24 hours, the transduced HepG2cells were co-cultured with 5×10⁵ PBMC cells and IL-2 in a round bottom96 well plate for 4 hours. The PBMC cells were pre-stimulated withzoledronic acid and IL-2 for 11 days to expand Vγ9Vδ2 T cells. Afterstaining for Vγ9Vδ2 and TNF-α using fluorophore-conjugated anti TCR-Vδ2and anti-TNF-α antibody, cells were analyzed via flow cytometry. Livecells were gated, and Vδ2+ and TNF-α+ cells were selected on a dot blot.The activated cytotoxic Vγ9Vδ2 T cells appeared in the upper rightquadrant of flow cytograms (FIG. 14, Panel B).

AAV Vector Construction.

FDPS shRNA sequence #4 (SEQ ID NO: 4) was inserted into the pAAV plasmid(Cell Biolabs). FDPS oligonucleotide sequences containing BamHI andEcoRI restriction sites were synthesized by Eurofins MWG Operon.Overlapping sense and antisense oligonucleotide sequences were mixed andannealed during cooling from 70 degrees Celsius to room temperature. ThepAAV was digested with the restriction enzymes BamHI and EcoRI for onehour at 37 degrees Celsius. The digested pAAV plasmid was purified byagarose gel electrophoresis and extracted from the gel using a DNA gelextraction kit from Thermo Scientific. The DNA concentrations weredetermined and vector to oligo (3:1 ratio) were mixed, allowed toanneal, and ligated. The ligation reaction was performed with T4 DNAligase for 30 minutes at room temperature. 2.5 microliters of theligation mix were added to 25 microliters of STBL3 competent bacterialcells. Transformation was achieved after heat-shock at 42 degreesCelsius. Bacterial cells were spread on agar plates containingampicillin and drug-resistant colonies (indicating the presence ofampicillin-resistance plasmids) were recovered and expanded in LB broth.To check for insertion of the oligo sequences, plasmid DNA was extractedfrom harvested bacteria cultures with the Thermo Scientific DNA miniprep kit. Insertion of shRNA sequences in the pAAV plasmid was verifiedby DNA sequencing using a specific primer for the promoter used toregulate shRNA expression. An exemplary AAV vector with a H1 promoter(SEQ ID NO: 16), shFDPS sequence (e.g., SEQ ID NO: 4), Left InvertedTerminal Repeat (Left ITR; SEQ ID NO: 65), and Right Inverted TerminalRepeat (Right ITR; SEQ ID NO: 66) can be found in FIG. 14, Panel A).

Production of AAV Particles.

The AAV-FDPS shRNA plasmid was combined with the plasmids pAAV-RC2 (CellBiolabs) and pHelper (Cell Biolabs). The pAAV-RC2 plasmid contains theRep and AAV2 capsid genes and pHelper contains the adenovirus E2A, E4,and VA genes. To produce AAV particles, these plasmids were transfectedin the ratio 1:1:1 (pAAV-shFDPS: pAAV-RC2: pHelper) into 293T cells. Fortransfection of cells in 150 mm dishes (BD Falcon), 10 micrograms ofeach plasmid were added together in 1 ml of DMEM. In another tube, 60microliters of the transfection reagent PEI (1 microgram/ml)(Polysciences) was added to 1 ml of DMEM. The two tubes were mixedtogether and allowed to incubate for 15 minutes. Then the transfectionmixture was added to cells and the cells were collected after 3 days.The cells were lysed by freeze/thaw lysis in dry ice/isopropanol.Benzonase nuclease (Sigma) was added to the cell lysate for 30 minutesat 37 degrees Celsius. Cell debris were then pelleted by centrifugationat 4 degrees Celsius for 15 minutes at 12,000 rpm. The supernatant wascollected and then added to target cells.

Example 13—Decreased RAP1 Prenylation in the Cells Transduced withLV-shFDPS and Treated with Zoledronic Acid

This Example illustrates that lentiviral-delivered shRNA targeting thehuman famesyl diphosphate synthase (FDPS) gene and zoledronic acidsynergize to inhibit famesyl diphosphate production.

FDPS is an enzyme in the isoprenoid synthesis pathway that catalyzes theproduction of famesyl diphosphate. Inhibiting the enzyme activity ofFDPS by zoledronic acid or reduced protein expression by shRNA-mediatedknock-down will result in reduced famesyl diphosphate levels.Famesylation of cellular proteins requires famesyl diphosphate. RAP1A isa protein that is modified by famesylation, which can be used as abiomarker for levels of cellular famesyl diphosphate. An antibody thatspecifically recognizes reduced RAP1A famesylation was used to measureFDPS activity after transduction with LV-shFDPS alone or in combinationwith zoledronic acid. HepG2 human hepatocellular carcinoma cells wereinfected with lentiviral vectors containing FDPS shRNA sequence #4. Forthe zoledronic acid treated cells, zoledronic acid (Sigma) was added forthe last 24 hours. After 48 hours, cells were lysed with NP-40 lysisbuffer and protein was quantified with the Bio-Rad protein assayreagent. Protein samples at 50 micrograms were electrophoresed on 4-12%Bis-Tris gels (Thermo Scientific and transferred to PVDF membranes (EMDMillipore). An immunoblot was performed using an anti-FDPS (ThermoScientific), anti-RAP1A (Santa Cruz), and an anti-actin (Sigma) antibodyas a protein loading control. Antibodies were bound with HRP-conjugatedsecondary antibodies and detected with a Licor c-DiGit Blot scannerusing the Immobilon Western ECL reagent (EMD Millipore). An increase inthe RAP1A band intensity correlates with reduced famesylation. RAP1Adefamesylation occurred only in the cells transduced with LV-shFDPS andtreated with zoledronic acid.

Example 14—Treatment of a Subject with Cancer

LV-FDPS is a Genetic Medicine Delivered by a Lentivirus Vector Via LocalAdministration to the Site of Late Stage, Non-Resectable HepatocellularCarcinoma

A Phase I clinical trial will test safety and feasibility of deliveringLV-FDPS to the site of hepatocellular carcinoma (HCC) using ultrasoundguided cannulation of the liver in patients without concomitantradiotherapy or chemotherapy. It is rationally predicted that this studywill result in the successful treatment of HCC. The study is an openlabel, 4×3 dose escalation (4 dose ranges, up to 3 subjects per dose) toidentify the maximum tolerable dose of LV-FDPS in patients 18 years orolder with Stage III/IV non-resectable HCC.

LV-FDPS is a genetic therapy designed to reduce expression in tumorcells of the enzyme famesyl diphosphate synthase. Experimental studiesshow that tumor cells modified by LV-FDPS induce the anti-tumor activityof human gamma delta T cells, including the capacity for tumor killingby cellular cytotoxicity.

Subjects with target lesions ≥1 cm in longest diameter (measured byhelical CT) and ≤4.9 cm maximum diameter and meeting inclusion andexclusion criteria detailed below, are enrolled into the next availabledosing category. A maximum of 3 subjects are recruited for each dosagegroup. The dose is number of transducing units of LV-FDPS as describedin the product release criteria, delivered via intrahepatic cannulationin a single bolus with volume not to exceed 25 mL. The minimum dose is1×10⁹ transducing units and escalation is 10-fold to a next dose of1×10¹⁰ transducing units, the next dose is 1×10¹¹ transducing units, anda maximum dose of 1×10¹² transducing units based on reported experiencewith recombinant adenovirus therapy for HCC (Sangro et al., A phase Iclinic trial of thymidine kinase-based gene therapy in advancedhepatocellular carcinoma, 2010, Cancer Gene Ther. 17:837-43). Subjectsare enrolled, treated and evaluated for 3 months. All safety evaluationsare completed for each group prior to enrolling and treating subjects atthe next higher dose level. Enrollment and dose escalation continueuntil a maximum tolerable dose is achieved or the study is terminated.

Cannulation is via the left subclavian artery until tip of catheter isat the proper hepatic artery junction. Cannulation is guided byultrasonography as described (Lin et al., Clinical effects ofintra-arterial infusion chemotherapy with cisplatin, mitomycin C,leucovor and 5-Fluorouracil for unresectable advanced hepatocellularcarcinoma, 2004, J Chin. Med. Assoc. 67:602-10).

Primary Outcome Measures

Safety: Systemic and locoregional adverse events are graded according toCTCAS and coded according to MedRA. The adverse events data for allsubjects at a single dose range will be evaluated prior to doseescalation. The final safety assessment incorporates data from all doseranges.

Secondary Outcome Measures

-   -   Lesion distribution and retention of LV-FDPS following        locoregional administration and subsequent biopsy or necropsy to        obtain tissues.    -   Objective response rate (ORR) in target and measurable non-local        lesions (if present) by physical analysis, medical imaging or        biopsy during 3 months after treatment.    -   Levels of LV-FDPS in blood stream during 10 minutes, 30 minutes,        1 hour and 1 day after local injection.    -   Changes in markers of hepatic function including ALP, ALT, ASAT,        total bilirubin and GGT during 3 months after treatment.    -   Disease free survival beyond historical control (no LV-FDPS)        patients in ad hoc analysis.

Inclusion Criteria

-   -   Greater than 18 years and including both males and females.    -   Diagnosis confirmed by histology or cytology or based on        currently accepted clinical standards of hepatocellular        carcinoma of parenchyma cell origin that is not amenable, at the        time of screening, to resection, transplant or other potentially        curative therapies.    -   Treating physician determines that the lesion is amenable to        locoregional targeted delivery.    -   Target lesion must represent measurable disease with a        unidimensional longest diameter of ≥1.0 cm by computed        tomography; the maximum longest diameter is ≤5.0 cm.    -   Kamofsky performance score 60-80% of ECOG values.    -   Life expectancy ≥12 weeks.    -   Hematopoietic function: WBC≥2,500/mm³; ANC≥1000/mm³; Hemoglobin        ≥8 g/dL; Platelet count ≥50,000/mm³; Coagulation INR≤1.3.    -   AST and ALT<5 times ULN; ALPS<5 time ULN. Bilirubin ≤1.5 times        ULV; Creatine ≤1.5 times ULN and eGFR≥50.    -   Thyroid function: Total T3 or free T3, total T4 or free T4 and        THC≤CTCAE Grade 2 abnormality.    -   Renal, cardiovascular and respiratory function adequate in the        opinion of the attending physician.    -   Immunological function: Circulating Vgamma9Vdelta2+ T cells        ≥30/mm³; no immunodeficiency disease.    -   Negative for HIV by serology and viral RNA test.    -   Written informed consent.

Exclusion Criteria

-   -   Target lesion contiguous with, encompasses or infiltrates blood        vessel.    -   Primary HCC amenable to resection, transplantation or other        potentially curative therapies.    -   Hepatic surgery or chemoembolization within the past 4 months.    -   Hepatic radiation or whole body radiation therapy within past 4        months.    -   Chemotherapy with 4 weeks or any use of nitrosourea, mitomycin C        or cisplatin.    -   Current or within past 4 weeks receipt of aminobisphosphonate        therapy    -   Investigational agents within 4 weeks or <5 drug half-lives.    -   Impaired wound healing due to diabetes.    -   Significant psychiatric illness, alcohol dependence or illicit        drug use.    -   Unwilling to comply with study protocols and reporting        requirements.    -   Aminobisphosphonate treatment within past 4 months.    -   Presence of clinically significant cardiovascular,        cerebrovascular (stroke), immunological (except hepatitis B or C        virus infection, viral hepatitis or cirrhosis), endocrine or        central nervous system disorders; current encephalopathy;        variceal bleeding requiring hospitalization or transfusion        within past 4 months.    -   History of HIV or acquired immune deficiency syndrome.    -   Current or prior treatment with antiretroviral medications.    -   Pregnant, lactating or refusal to adopt barrier or chemical        contraceptive use throughout trial and follow-up interval.        LV-FDPS is a Genetic Medicine Delivered by a Lentivirus Vector        Via Local Administration to the Site of Late Stage,        Non-Resectable Hepatocellular Carcinoma—Adjunct Administration        of Aminobisphosphonate

A Phase I clinical trial will test safety and feasibility of deliveringLV-FDPS to the site of hepatocellular carcinoma (HCC) using ultrasoundguided cannulation of the liver in patients with concomitantaminobisphosphonate chemotherapy. It is rationally predicted that thisstudy will result in the successful treatment of HCC. The study is anopen label, 4×3 dose escalation (4 dose ranges, up to 3 subjects perdose) to identify the maximum tolerable dose of LV-FDPS in patients 18years or older with Stage III/IV non-resectable HCC.

LV-FDPS is a genetic therapy designed to reduce expression in tumorcells of the enzyme famesyl diphosphate synthase. Experimental studiesshow that tumor cells modified by LV-FDPS induce the anti-tumor activityof human gamma delta T cells, including the capacity for tumor killingby cellular cytotoxicity. Prior experimental studies also showed thepotential for positive interactions of LV-FDPS and specificaminobisphosphonate drugs that may be prescribed in primary ormetastatic diseases. For this study, subjects will receive doseescalating amounts of LV-FDPS with continuous standard of care dosingwith Aredia® (pamidronate), Zometa® (zoledronic acid) or Actonel®(risedronate) according to physician advice and subject preference.

Subjects with target lesions ≥1 cm in longest diameter (measured byhelical CT) and ≤4.9 cm maximum diameter and meeting inclusion andexclusion criteria detailed below, are enrolled and started onaminobisphosphonate therapy. 30 days later size of the target lesion isre-evaluated to ensure subjects still meet starting criteria forLV-FDPS. Subjects without objective clinical response onaminobisphosphonate are enrolled into the next available LV-FDPS dosingcategory. A maximum of 3 subjects are recruited for each dosage groupand all continue on aminobisphosphonate fir the study duration unlessotherwise advised by the attending physician. The LV-FDPS dose is anumber of transducing units of LV-FDPS as described in the productrelease criteria, delivered via intrahepatic cannulation in a singlebolus with volume not to exceed 25 mL. The minimum dose is 1×10⁹transducing units and escalation is 10-fold to a next dose of 1×10¹⁰transducing units, the next dose is 1×10¹¹ transducing units, and amaximum dose of 1×10¹² transducing units based on reported experiencewith recombinant adenovirus therapy for HCC (Sangro, et al., A phase Iclinic trial of thymidine kinase-based gene therapy in advancedhepatocellular carcinoma, 2010, Cancer Gene Ther. 17:837-43). Subjectsare enrolled, treated and evaluated for 3 months. All safety evaluationsare completed for each group prior to enrolling and treating subjects atthe next higher dose level. Enrollment and dose escalation continueuntil a maximum tolerable dose is achieved or the study is terminated.

Cannulation is via the left subclavian artery until tip of catheter isat the proper hepatic artery junction. Cannulation is guided byultrasonography as described (Lin et al., Clinical effects ofintra-arterial infusion chemotherapy with cisplatin, mitomycin C,leucovor and 5-Fluorouracil for unresectable advanced hepatocellularcarcinoma, 2004, J Chin. Med. Assoc. 67:602-10).

Primary Outcome Measures

Safety: Systemic and locoregional adverse events are graded according toCTCAS and coded according to MedRA. The adverse events data for allsubjects at a single dose range will be evaluated prior to doseescalation. The final safety assessment incorporates data from all doseranges.

Secondary Outcome Measures

-   -   Lesion distribution and retention of LV-FDPS following        locoregional administration and subsequent biopsy or necropsy to        obtain tissues.    -   Objective response rate (ORR) in target and measurable non-local        lesions (if present) by physical analysis, medical imaging or        biopsy during 3 months after treatment.    -   Levels of LV-FDPS in blood stream during 10 minutes, 30 minutes,        1 hour and 1 day after local injection.    -   Changes in markers of hepatic function including ALP, ALT, ASAT,        total bilirubin and GGT during 3 months after treatment.    -   Disease free survival beyond historical control (no LV-FDPS)        patients in ad hoc analysis.

Inclusion Criteria

-   -   Greater than 18 years and including both males and females.    -   Diagnosis confirmed by histology or cytology or based on        currently accepted clinical standards of hepatocellular        carcinoma of parenchyma cell origin that is not amenable, at the        time of screening, to resection, transplant or other potentially        curative therapies.    -   Treating physician determines that the lesion is amenable to        locoregional targeted delivery.    -   Target lesion must represent measurable disease with a        unidimensional longest diameter of ≥1.0 cm by computed        tomography; the maximum longest diameter is ≤5.0 cm.    -   Kamofsky performance score 60-80% of ECOG values.    -   Life expectancy ≥12 weeks.    -   Hematopoietic function: WBC≥2,500/mm³; ANC≥1000/mm³; Hemoglobin        ≥8 g/dL; Platelet count ≥50,000/mm³; Coagulation INR≤1.3.    -   AST and ALT<5 times ULN; ALPS<5 time ULN. Bilirubin ≤1.5 times        ULV; Creatine ≤1.5 times ULN and eGFR≥50.    -   Thyroid function: Total T3 or free T3, total T4 or free T4 and        THC≤CTCAE Grade 2 abnormality.    -   Renal, cardiovascular and respiratory function adequate in the        opinion of the attending physician.    -   Immunological function: Circulating Vgamma9Vdelta2+ T cells        ≥30/mm³; no immunodeficiency disease.    -   Negative for HIV by serology and viral RNA test.    -   Written informed consent.

Exclusion Criteria

-   -   Intolerant to or unwilling to continue aminobisphosphonate        adjunct therapy.    -   Objective clinical response after aminobisphosphonate therapy.    -   Target lesion contiguous with, encompasses or infiltrates blood        vessel.    -   Primary HCC amenable to resection, transplantation or other        potentially curative therapies.    -   Hepatic surgery or chemoembolization within the past 4 months.    -   Hepatic radiation or whole body radiation therapy within past 4        months.    -   Chemotherapy excluding aminobisphosphonate, within 4 weeks or        any use of nitrosourea, mitomycin C or cisplatin.    -   Investigational agents within 4 weeks or <5 drug half-lives.    -   Impaired wound healing due to diabetes.    -   Significant psychiatric illness, alcohol dependence or illicit        drug use.    -   Unwilling to comply with study protocols and reporting        requirements.    -   Presence of clinically significant cardiovascular,        cerebrovascular (stroke), immunological (except hepatitis B or C        virus infection, viral hepatitis or cirrhosis), endocrine or        central nervous system disorders; current encephalopathy;        variceal bleeding requiring hospitalization or transfusion        within past 4 months.    -   History of HIV or acquired immune deficiency syndrome.    -   Current or prior treatment with antiretroviral medications.    -   Pregnant, lactating or refusal to adopt barrier or chemical        contraceptive use throughout trial and follow-up interval.

Example 15—Treatment of a Subject with Chronic Viral Disease(s) of theLiver

LV-FDPS is a Genetic Medicine Delivered by a Lentivirus Vector Via LocalAdministration to Liver for the Treatment of Hepatitis B Virus,Hepatitis C Virus, HIV or Other Viral Infection of the Liver

A Phase I clinical trial will test safety and feasibility of deliveringLV-FDPS to virally infected liver using ultrasound guided cannulation.It is rationally predicted that this study will result in the successfultreatment of infections of the liver. The study is an open label, 4×3dose escalation (4 dose ranges, up to 3 subjects per dose) to identifythe maximum tolerable dose of LV-FDPS in patients 18 years or older withchronic viral disease of the liver that is resistant to chemotherapy.

LV-FDPS is a genetic therapy designed to reduce expression in tumorcells of the enzyme famesyl diphosphate synthase. Experimental studiesshow that tumor cells modified by LV-FDPS induce human gamma delta Tcells, including a capacity for cellular cytotoxicity againstvirally-infected cells.

Subjects with confirmed viral infection of the liver including hepatitisB virus, hepatitis C virus, HIV or other viruses are enrolled into thenext available LV-FDPS dosing category. A maximum of 3 subjects arerecruited for each dosage group. The LV-FDPS dose is a number oftransducing units of LV-FDPS as described in the product releasecriteria, delivered via intrahepatic cannulation in a single bolus withvolume not to exceed 25 mL. The minimum dose is 1×10⁹ transducing unitsand escalation is 10-fold to a next dose of 1×10¹⁰ transducing units,the next dose is 1×10¹¹ transducing units, and a maximum dose of 1×10¹²transducing units based on reported experience with recombinantadenovirus therapy for HCC (Sangro, et al., A phase I clinic trial ofthymidine kinase-based gene therapy in advanced hepatocellularcarcinoma, 2010, Cancer Gene Ther. 17:837-43). Subjects are enrolled,treated and evaluated for 3 months. All safety evaluations are completedfor each group prior to enrolling and treating subjects at the nexthigher dose level. Enrollment and dose escalation continue until amaximum tolerable dose is achieved or the study is terminated.

Cannulation is via the left subclavian artery until tip of catheter isat the proper hepatic artery junction. Cannulation is guided byultrasonography as described (Lin et al., Clinical effects ofintra-arterial infusion chemotherapy with cisplatin, mitomycin C,leucovor and 5-Fluorouracil for unresectable advanced hepatocellularcarcinoma, 2004, J. Chin. Med. Assoc. 67:602-10).

Primary Outcome Measures

Safety: Systemic and locoregional adverse events are graded according toCTCAS and coded according to MedRA. The adverse events data for allsubjects at a single dose range will be evaluated prior to doseescalation. The final safety assessment incorporates data from all doseranges.

Secondary Outcome Measures

-   -   Lesion distribution and retention of LV-FDPS following        locoregional administration and subsequent biopsy or necropsy to        obtain tissues.    -   Objective response rate (ORR) measured as a Sustained Viral        Response (SVR) within the organ or systemically during 3 months        after treatment.    -   Levels of LV-FDPS in blood stream during 10 minutes, 30 minutes,        1 hour and 1 day after local injection.    -   Changes in markers of hepatic function including ALP, ALT, ASAT,        total bilirubin and GGT during 3 months after treatment.    -   Disease free survival beyond historical control (no LV-FDPS)        patients in ad hoc analysis.

Inclusion Criteria

-   -   Greater than 18 years and including both males and females.    -   Diagnosis confirmed by histology or cytology or based on        currently accepted clinical standards of chronic viral infection        of the liver that is not amenable, at the time of screening, to        resection, transplant or other potentially curative therapies.    -   Treating physician determines that the lesion is amenable to        locoregional targeted delivery.    -   Kamofsky performance score 60-80% of ECOG values.    -   Life expectancy ≥12 weeks.    -   Hematopoietic function: WBC≥2,500/mm³; ANC≥1000/mm³; Hemoglobin        ≥8 g/dL; Platelet count ≥50,000/mm³; Coagulation INR≤1.3.    -   AST and ALT<5 times ULN; ALPS<5 time ULN. Bilirubin ≤1.5 times        ULV; Creatine ≤1.5 times ULN and eGFR≥50.    -   Thyroid function: Total T3 or free T3, total T4 or free T4 and        THC≤CTCAE Grade 2 abnormality.    -   Renal, cardiovascular and respiratory function adequate in the        opinion of the attending physician.    -   Immunological function: Circulating Vgamma9Vdelta2+ T cells        ≥30/mm³; no immunodeficiency disease.    -   Negative for HIV by serology and viral RNA test.    -   Written informed consent.

Exclusion Criteria

-   -   Chronic viral disease amenable to resection, transplantation or        other potentially curative therapies.    -   Hepatic surgery or chemoembolization within the past 4 months.    -   Hepatic radiation or whole body radiation therapy within past 4        months.    -   Investigational agents within 4 weeks or <5 drug half-lives.    -   Current (within past 4 weeks) or ongoing receipt of        aminobisphosphonate therapy.    -   Impaired wound healing due to diabetes.    -   Significant psychiatric illness, alcohol dependence or illicit        drug use.    -   Unwilling to comply with study protocols and reporting        requirements.    -   Presence of clinically significant cardiovascular,        cerebrovascular (stroke), immunological (except virus infection,        viral hepatitis or cirrhosis), endocrine or central nervous        system disorders; current encephalopathy; variceal bleeding        requiring hospitalization or transfusion within past 4 months.    -   Pregnant, lactating or refusal to adopt barrier or chemical        contraceptive use throughout trial and follow-up interval.        LV-FDPS is a Genetic Medicine Delivered by a Lentivirus Vector        Via Local Administration to Liver for the Treatment of Hepatitis        B Virus, Hepatitis C Virus, HIV or Other Viral Infection of the        Liver—Concomitant Adjunct Aminobisphosphonate Therapy

A Phase I clinical trial will test safety and feasibility of deliveringLV-FDPS to virally infected liver using ultrasound guided cannulation.It is rationally predicted that this study will result in the successfultreatment of infections of the liver. The study is an open label, 4×3dose escalation (4 dose ranges, up to 3 subjects per dose) to identifythe maximum tolerable dose of LV-FDPS in patients 18 years or older withchronic viral disease of the liver that is resistant to chemotherapy.

LV-FDPS is a genetic therapy designed to reduce expression in tumorcells of the enzyme farnesyl diphosphate synthase. Experimental studiesshow that tumor cells modified by LV-FDPS induce human gamma delta Tcells, including a capacity for cellular cytotoxicity againstvirally-infected cells. Prior experimental studies also showed thepotential for positive interactions of LV-FDPS and specificaminobisphosphonate drugs that may be prescribed during infectiousdisease. For this study, subjects will receive dose escalating amountsof LV-FDPS with continuous standard of care dosing with Aredia®(pamidronate), Zometa® (zoledronic acid) or Actonel® (risedronate)according to physician advice and subject preference.

Subjects with confirmed viral infection of the liver including hepatitisB virus, hepatitis C virus, HIV or other viruses will initiateaminobisphosphonate therapy for 45 days before re-screening to meetenrollment criteria for LV-FDPS treatment of infectious disease.Eligible subjects are enrolled into the next available LV-FDPS dosingcategory. A maximum of 3 subjects are recruited for each dosage group.The LV-FDPS dose is a number of transducing units of LV-FDPS asdescribed in the product release criteria, delivered via intrahepaticcannulation in a single bolus with volume not to exceed 25 mL. Theminimum dose is 1×10⁹ transducing units and escalation is 10-fold to anext dose of 1×10¹⁰ transducing units, the next dose is 1×10¹¹transducing units, and a maximum dose of 1×10¹² transducing units basedon reported experience with recombinant adenovirus therapy for HCC(Sangro, et al., A phase I clinic trial of thymidine kinase-based genetherapy in advanced hepatocellular carcinoma, 2010, Cancer Gene Ther.17:837-43). Subjects are enrolled, treated and evaluated for 3 months.All safety evaluations are completed for each group prior to enrollingand treating subjects at the next higher dose level. Enrollment and doseescalation continue until a maximum tolerable dose is achieved or thestudy is terminated.

Cannulation is via the left subclavian artery until tip of catheter isat the proper hepatic artery junction. Cannulation is guided byultrasonography as described (Lin et al., Clinical effects ofintra-arterial infusion chemotherapy with cisplatin, mitomycin C,leucovor and 5-Fluorouracil for unresectable advanced hepatocellularcarcinoma, 2004, J Chin. Med. Assoc. 67:602-10).

Primary Outcome Measures

Safety: Systemic and locoregional adverse events are graded according toCTCAS and coded according to MedRA. The adverse events data for allsubjects at a single dose range will be evaluated prior to doseescalation. The final safety assessment incorporates data from all doseranges.

Secondary Outcome Measures

-   -   Lesion distribution and retention of LV-FDPS following        locoregional administration and subsequent biopsy or necropsy to        obtain tissues.    -   Objective response rate (ORR) measured as a Sustained Viral        Response (SVR) within the organ or systemically during 3 months        after treatment.    -   Levels of LV-FDPS in blood stream during 10 minutes, 30 minutes,        1 hour and 1 day after local injection.    -   Changes in markers of hepatic function including ALP, ALT, ASAT,        total bilirubin and GGT during 3 months after treatment.    -   Disease free survival beyond historical control (no LV-FDPS)        patients in ad hoc analysis.

Inclusion Criteria

-   -   Greater than 18 years and including both males and females.    -   Diagnosis confirmed by histology or cytology or based on        currently accepted clinical standards of chronic viral infection        of the liver that is not amenable, at the time of screening, to        resection, transplant or other potentially curative therapies.    -   Treating physician determines that the lesion is amenable to        locoregional targeted delivery.    -   Kamofsky performance score 60-80% of ECOG values.    -   Life expectancy ≥12 weeks.    -   Hematopoietic function: WBC≥2,500/mm³; ANC≥1000/mm³; Hemoglobin        ≥8 g/dL; Platelet count ≥50,000/mm³; Coagulation INR≤1.3.    -   AST and ALT<5 times ULN; ALPS<5 time ULN. Bilirubin ≤1.5 times        ULV; Creatine ≤1.5 times ULN and eGFR≥50.    -   Thyroid function: Total T3 or free T3, total T4 or free T4 and        THC≤CTCAE Grade 2 abnormality.    -   Renal, cardiovascular and respiratory function adequate in the        opinion of the attending physician.    -   Immunological function: Circulating Vgamma9Vdelta2+ T cells        ≥30/mm³; no immunodeficiency disease.    -   Negative for HIV by serology and viral RNA test.    -   Written informed consent.

Exclusion Criteria

-   -   Chronic viral disease amenable to resection, transplantation or        other potentially curative therapies.    -   Hepatic surgery or chemoembolization within the past 4 months.    -   Hepatic radiation or whole body radiation therapy within past 4        months.    -   Investigational agents within 4 weeks or <5 drug half-lives.    -   Impaired wound healing due to diabetes.    -   Significant psychiatric illness, alcohol dependence or illicit        drug use.    -   Unwilling to comply with study protocols and reporting        requirements.    -   Presence of clinically significant cardiovascular,        cerebrovascular (stroke), immunological (except virus infection,        viral hepatitis or cirrhosis), endocrine or central nervous        system disorders; current encephalopathy; variceal bleeding        requiring hospitalization or transfusion within past 4 months.    -   Pregnant, lactating or refusal to adopt barrier or chemical        contraceptive use    -   throughout trial and follow-up interval.        Sequences

The following sequences are referred to herein:

SEQ ID NO: Description Sequence 1 FDPS shRNAGTCCTGGAGTACAATGCCATTCTCGAGAATGGCATT sequence #1 GTACTCCAGGACTTTTT 2FDPS shRNA GCAGGATTTCGTTCAGCACTTCTCGAGAAGTGCTGA sequence #2ACGAAATCCTGCTTTTT 3 FDPS shRNA GCCATGTACATGGCAGGAATTCTCGAGAATTCCTGCsequence #3 CATGTACATGGCTTTTT 4 FDPS shRNAGCAGAAGGAGGCTGAGAAAGTCTCGAGACTTTCTC sequence #4 AGCCTCCTTCTGCTTTTT 5miR30 FDPS AAGGTATATTGCTGTTGACAGTGAGCGACACTTTCT sequence #1CAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAA GGAGGCTGAGAAAGTGCTGCCTACTGCCTCGGACTTCAAGGGGCT 6 miR30 FDPS AAGGTATATTGCTGTTGACAGTGAGCGACACTTTCT sequence #2CAGCCTCCTTCTGCGTGAAGCCACAGATGGCAGAA GGGCTGAGAAAGTGCTGCCTACTGCCTCGGACTTCAAGGGGCT 7 miR30 FDPS TGCTGTTGACAGTGAGCGACTTTCTCAGCCTCCTTCT sequence #3GCGTGAAGCCACAGATGGCAGAAGGAGGCTGAGAA AGTTGCCTACTGCCTCGGA 8 miR155 FDPSCCTGGAGGCTTGCTGAAGGCTGTATGCTGACTTTCT sequence #1CAGCCTCCTTCTGCTTTTGGCCACTGACTGAGCAGA AGGGCTGAGAAAGTCAGGACACAAGGCCTGTTACTAGCACTCA 9 miR21 FDPS CATCTCCATGGCTGTACCACCTTGTCGGGACTTTCTC sequence #1AGCCTCCTTCTGCCTGTTGAATCTCATGGCAGAAGGAGGCGAGAAAGTCTGACATTTTGGTATCTTTCATCT GACCA 10 miR185 FDPSGGGCCTGGCTCGAGCAGGGGGCGAGGGATACTTTC sequence #1TCAGCCTCCTTCTGCTGGTCCCCTCCCCGCAGAAGGAGGCTGAGAAAGTCCTTCCCTCCCAATGACCGCGTC TTCGTCG 11 Rous SarcomaGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACAT virus (RSV)GGTAACGATGAGTTAGCAACATGCCTTACAAGGAG promoterAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTA AGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAAT TGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAATAAACG 12 5′ Long terminalGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGC repeat (LTR)TCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA 13 Psi PackagingTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGG signal AGAGAG 14 Rev responseAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGG element (RRE)AAGCACTATGGGCGCAGCCTCAATGACGCTGACGG TACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAA CAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATA CCTAAAGGATCAACAGCTCC 15 CentralTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTG polypurine tractCAGGGGAAAGAATAGTAGACATAATAGCAACAGAC (cPPT)ATACAAACTAAAGAATTACAAAAACAAATTACAAA ATTCAAAATTTTA 16 Polymerase IIIGAACGCTGACGTCATCAACCCGCTCCAAGGAATCG shRNACGGGCCCAGTGTCACTAGGCGGGAACACCCAGCGC promoters; H1GCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGAC promoterAGGGGAGTGGCGCCCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACC ACTT 17 Long WPREAATCAACCTCTGATTACAAAATTTGTGAAAGATTGA sequenceCTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCT CCCCGCCT 18 3′ delta LTRTGGAAGGGCTAATTCACTCCCAACGAAGATAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAG TGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCA19 Helper/Rev; GCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTG Chicken betaCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCA actin (CAG)ATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGC promoter;GATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGG TranscriptionCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCG AGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCG GCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG 20 Helper/Rev; HIV ATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGA Gag; ViralATTAGATCGATGGGAAAAAATTCGGTTAAGGCCAG capsidGGGGAAAGAAAAAATATAAATTAAAACATATAGTA TGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACA AATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAA AAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAG CAGCAGCTGACACAGGACACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCA AATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGTAGAAGAGAAGGCTTTCA GCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTA AACACAGTGGGGGGACATCAAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGAAT GGGATAGAGTGCATCCAGTGCATGCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAAGGGGAAGTGA CATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGGATGGATGACACATAATCCACCTATCCCAGTAG GAGAAATCTATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCATT CTGGACATAAGACAAGGACCAAAGGAACCCTTTAGAGACTATGTAGACCGATTCTATAAAACTCTAAGAGC CGAGCAAGCTTCACAAGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGTCCAAAATGCGAACCCAGATT GTAAGACTATTTTAAAAGCATTGGGACCAGGAGCGACACTAGAAGAAATGATGACAGCATGTCAGGGAGT GGGGGGACCCGGCCATAAAGCAAGAGTTTTGGCTGAAGCAATGAGCCAAGTAACAAATCCAGCTACCATA ATGATACAGAAAGGCAATTTTAGGAACCAAAGAAAGACTGTTAAGTGTTTCAATTGTGGCAAAGAAGGGCA CATAGCCAAAAATTGCAGGGCCCCTAGGAAAAAGGGCTGTTGGAAATGTGGAAAGGAAGGACACCAAATG AAAGATTGTACTGAGAGACAGGCTAATTTTTTAGGGAAGATCTGGCCTTCCCACAAGGGAAGGCCAGGGAA TTTTCTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGAAGAGAGCTTCAGGTTTGGGGAAGAGACAACAA CTCCCTCTCAGAAGCAGGAGCCGATAGACAAGGAACTGTATCCTTTAGCTTCCCTCAGATCACTCTTTGGCA GCGACCCCTCGTCACAATAA 21Helper/Rev; HIV ATGAATTTGCCAGGAAGATGGAAACCAAAAATGATPol; Protease and reverse AGGGGGAATTGGAGGTTTTATCAAAGTAGGACAGTtranscriptase ATGATCAGATACTCATAGAAATCTGCGGACATAAAGCTATAGGTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGCTGCACTTTAAATTTTCCCATTAGTCCTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCA AAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAAATGGAAA AGGAAGGAAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAA GACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGGGAAG TTCAATTAGGAATACCACATCCTGCAGGGTTAAAACAGAAAAAATCAGTAACAGTACTGGATGTGGGCGAT GCATATTTTTCAGTTCCCTTAGATAAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTT CCACAGGGATGGAAAGGATCACCAGCAATATTCCAGTGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTCATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCA GCATAGAACAAAAATAGAGGAACTGAGACAACATCTGTTGAGGTGGGGATTTACCACACCAGACAAAAAA CATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATA GTGCTGCCAGAAAAGGACAGCTGGACTGTCAATGACATACAGAAATTAGTGGGAAAATTGAATTGGGCAA GTCAGATTTATGCAGGGATTAAAGTAAGGCAATTATGTAAACTTCTTAGGGGAACCAAAGCACTAACAGAA GTAGTACCACTAACAGAAGAAGCAGAGCTAGAACTGGCAGAAAACAGGGAGATTCTAAAAGAACCGGTAC ATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCAAGGCCAATGGACA TATCAAATTTATCAAGAGCCATTTAAAAATCTGAAAACAGGAAAATATGCAAGAATGAAGGGTGCCCACAC TAATGATGTGAAACAATTAACAGAGGCAGTACAAAAAATAGCCACAGAAAGCATAGTAATATGGGGAAAG ACTCCTAAATTTAAATTACCCATACAAAAGGAAACATGGGAAGCATGGTGGACAGAGTATTGGCAAGCCAC CTGGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCTTAGTGAAGTTATGGTACCAGTTAGAGAAAGAAC CCATAATAGGAGCAGAAACTTTCTATGTAGATGGGGCAGCCAATAGGGAAACTAAATTAGGAAAAGCAGG ATATGTAACTGACAGAGGAAGACAAAAAGTTGTCCCCCTAACGGACACAACAAATCAGAAGACTGAGTTA CAAGCAATTCATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTGACAGACTCACAATATGCATT GGGAATCATTCAAGCACAACCAGATAAGAGTGAATCAGAGTTAGTCAGTCAAATAATAGAGCAGTTAATA AAAAAGGAAAAAGTCTACCTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAAGTAGATG GGTTGGTCAGTGCTGGAATCAGGAAAGTACTA 22Helper Rev; HIV TTTTTAGATGGAATAGATAAGGCCCAAGAAGAACA Integras e;TGAGAAATATCACAGTAATTGGAGAGCAATGGCTA Integration ofGTGATTTTAACCTACCACCTGTAGTAGCAAAAGAAA viral RNATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGG GAAGCCATGCATGGACAAGTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAA AAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGG CAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACAGACAATGG CAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTAT GAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAA ATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAG TAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGG GTTTATTACAGGGACAGCAGAGATCCAGTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGG CAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATCAGGGATTA TGGAAAACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGGATTAA 23 Helper/Rev; HIVAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGG RRE; Binds RevAAGCACTATGGGCGCAGCGTCAATGACGCTGACGG elementTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGC AGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAG CAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCT 24 Helper/Rev; HIVATGGCAGGAAGAAGCGGAGACAGCGACGAAGAAC Rev; NuclearTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATC export andAAAGCAACCCACCTCCCAATCCCGAGGGGACCCGA stabilize viralCAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAG mRNAAGAGACAGAGACAGATCCATTCGATTAGTGAACGG ATCCTTAGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACG CAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAATAG 25 Envelope; CMVACATTGATTATTGACTAGTTATTAATAGTAATCAAT promoter;TACGGGGTCATTAGTTCATAGCCCATATATGGAGTT TranscriptionCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAAT GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGC 26 Envelope; VSV- ATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGG; Glycoprotein GGGTGAATTGCAAGTTCACCATAGTTTTTCCACACA envelope-cellACCAAAAAGGAAACTGGAAAAATGTTCCTTCTAATT entryACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGC ATAATGACTTAATAGGCACAGCCTTACAAGTCAAAATGCCCAAGAGTCACAAGGCTATTCAAGCAGACGG TTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCCATCCGATCCTTCACTCCATCTGTAGAACAATG CAAGGAAAGCATTGAACAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGTTGTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTGACTCCTCACCATGTGCTGGTTGATGAATACA CAGGAGAATGGGTTGATTCACAGTTCATCAACGGAAAATGCAGCAATTACATATGCCCCACTGTCCATAACTCTACAACCTGGCATTCTGACTATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCATGGACATCACCTTCTTCTCAGAGGACGGAGAGCTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAGAAGTAACTACTTTGCTT ATGAAACTGGAGGCAAGGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCTCAGACCTCAGTGGATGTAAGTCTAATTCAGGACGTTGAGAGGATCTTGGATTATTCCCTC TGCCAAGAAACCTGGAGCAAAATCAGAGCGGGTCTTCCAATCTCTCCAGTGGATCTCAGCTATCTTGCTCCTAAAAACCCAGGAACCGGTCCTGCTTTCACCATAATCAATGGTACCCTAAAATACTTTGAGACCAGATACATCAGAGTCGATATTGCTGCTCCAATCCTCTCAAGAATG GTCGGAATGATCAGTGGAACTACCACAGAAAGGGAACTGTGGGATGACTGGGCACCATATGAAGACGTGG AAATTGGACCCAATGGAGTTCTGAGGACCAGTTCAGGATATAAGTTTCCTTTATACATGATTGGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTCAAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTATTTTTTGGTGATACTGGGCTATCCAAAAATCCAATCGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTTCTCCGAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAAAGACAGATTTATACAGA CATAGAGATGA 27 Helper/Rev;TAGTTATTAATAGTAATCAATTACGGGGTCATTAGT CMV earlyTCATAGCCCATATATGGAGTTCCGCGTTACATAACT (CAG) enhancer;TACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG EnhanceACCCCCGCCCATTGACGTCAATAATGACGTATGTTC TranscriptionCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTA CTTGGCAGTACATCTACGTATTAGTCATC 28Helper/Rev; GGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTC Chicken betaCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTG actin intron;ACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGG Enhance geneCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAA expressionTGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGC CTTAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGC GTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTG TGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGG GAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTG TAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGCGG GGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCC GCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGG CGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGGC GGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCGAAGCGGTGCGGCGCCGGCA GGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCATCTCCAGCCTCGG GGCTGCCGCAGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGAC CGGCGG 29 Helper/Rev;AGATCTTTTTCCCTCTGCCAAAAATTATGGGGACAT Rabbit betaCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATA globin poly A;AAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAA RNA stabilityTTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCATATGCTGGCTGCC ATGAACAAAGGTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTC TCTTATGAAGATC 30 Envelope; BetaGTGAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCG globin intron;CTATTGTAAAATTCATGTTATATGGAGGGGGCAAAG Enhance geneTTTTCAGGGTGTTGTTTAGAATGGGAAGATGTCCCT expressionTGTATCACCATGGACCCTCATGATAATTTTGTTTCTTTCACTTTCTACTCTGTTGACAACCATTGTCTCCTCTTATTTTCTTTTCATTTTCTGTAACTTTTTCGTTAAACTTTAGCTTGCATTTGTAACGAATTTTTAAATTCACTTTTGTTTATTTGTCAGATTGTAAGTACTTTCTCTAATCACTTTTTTTTCAAGGCAATCAGGGTATATTATATTGTACTTCAGCACAGTTTTAGAGAACAATTGTTATAATTAAATGATAAGGTAGAATATTTCTGCATATAAATTCTGGCTGGCGTGGAAATATTCTTATTGGTAGAAACAACTACACCCTGGTCATCATCCTGCCTTTCTCTTTATGGTTACAATGATATACACTGTTTGAGATGAGGATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGCTAACCATGTT CATGCCTTCTTCTCTTTCCTACAG 31Envelope; Rabbit AGATCTTTTTCCCTCTGCCAAAAATTATGGGGACAT beta globin polyCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATA A; RNA stabilityAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGC CATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCT TCTCTTATGGAGATC 32 PrimerTAAGCAGAATTCATGAATTTGCCAGGAAGAT 33 PrimerCCATACAATGAATGGACACTAGGCGGCCGCACGAA T 34 Gag, Pol,GAATTCATGAATTTGCCAGGAAGATGGAAACCAAA IntegraseAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAA fragmentGACAGTATGATCAGATACTCATAGAAATCTGCGGA CATAAAGCTATAGGTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGCTGCACTTTAAATTTTCCCATTAGTCCTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGAT GGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAAA TGGAAAAGGAAGGAAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAG AAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGGGAAGTTCAATTAGGAATACCACATCCTGCAGGGTT AAAACAGAAAAAATCAGTAACAGTACTGGATGTGGGCGATGCATATTTTTCAGTTCCCTTAGATAAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAA ACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAAT ATTCCAGTGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTCATCTATCAAT ACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAACTGAGA CAACATCTGTTGAGGTGGGGATTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAG CCTATAGTGCTGCCAGAAAAGGACAGCTGGACTGTCAATGACATACAGAAATTAGTGGGAAAATTGAATT GGGCAAGTCAGATTTATGCAGGGATTAAAGTAAGGCAATTATGTAAACTTCTTAGGGGAACCAAAGCACTA ACAGAAGTAGTACCACTAACAGAAGAAGCAGAGCTAGAACTGGCAGAAAACAGGGAGATTCTAAAAGAAC CGGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCAAGGCCAA TGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTGAAAACAGGAAAGTATGCAAGAATGAAGGGTGC CCACACTAATGATGTGAAACAATTAACAGAGGCAGTACAAAAAATAGCCACAGAAAGCATAGTAATATGG GGAAAGACTCCTAAATTTAAATTACCCATACAAAAGGAAACATGGGAAGCATGGTGGACAGAGTATTGGC AAGCCACCTGGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCTTAGTGAAGTTATGGTACCAGTTAGAGA AAGAACCCATAATAGGAGCAGAAACTTTCTATGTAGATGGGGCAGCCAATAGGGAAACTAAATTAGGAAA AGCAGGATATGTAACTGACAGAGGAAGACAAAAAGTTGTCCCCCTAACGGACACAACAAATCAGAAGACT GAGTTACAAGCAATTCATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTGACAGACTCACAATA TGCATTGGGAATCATTCAAGCACAACCAGATAAGAGTGAATCAGAGTTAGTCAGTCAAATAATAGAGCAG TTAATAAAAAAGGAAAAAGTCTACCTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAAG TAGATAAATTGGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAGATGGAATAGATAAGGCCCAAGAAGA ACATGAGAAATATCACAGTAATTGGAGAGCAATGGCTAGTGATTTTAACCTACCACCTGTAGTAGCAAAAG AAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGGGAAGCCATGCATGGACAAGTAGACTGTAGCCC AGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTG GATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCA GGAAGATGGCCAGTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGC CTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAAT CTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGT ACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGA ATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATT TTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAA GGGGCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATCAGGG ATTATGGAAAACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGGATTAA 35 DNA FragmentTCTAGAATGGCAGGAAGAAGCGGAGACAGCGACGA containing Rev,AGAGCTCATCAGAACAGTCAGACTCATCAAGCTTCT RRE and rabbitCTATCAAAGCAACCCACCTCCCAATCCCGAGGGGA beta globin polyCCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGG AAGAGAGAGACAGAGACAGATCCATTCGATTAGTGA ACGGATCCTTGGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGG GACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAATAGAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCA GGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCA GCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCA AGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTA AAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCATATGCTGGCTGCCATGAACAAAGGTG GCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGAAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGT GTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAAT GGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCAGCGGCCG CCCCGGG 36 DNA fragmentACGCGTTAGTTATTAATAGTAATCAATTACGGGGTC containing theATTAGTTCATAGCCCATATATGGAGTTCCGCGTTAC CAGATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC enhancer/promoter/CAACGACCCCCGCCCATTGACGTCAATAATGACGTA intronTGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG sequenceACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCG ATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGA GGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGG CGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTG ACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCTTTGTGCG GGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCC CGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGC CGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGG GGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGC GGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGG CCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGC GGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTG GCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCGAAGCGGTGCGGCGCCGG CAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCATCTCCAGCCTC GGGGCTGCCGCAGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTG ACCGGCGGGAATTC 37 DNA fragmentGAATTCATGAAGTGCCTTTTGTACTTAGCCTTTTTAT containing VSV-TCATTGGGGTGAATTGCAAGTTCACCATAGTTTTTC GCACACAACCAAAAAGGAAACTGGAAAAATGTTCCT TCTAATTACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGCATAATGACTTAATAGGCACAGCCTTACAA GTCAAAATGCCCAAGAGTCACAAGGCTATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCCATCCGATCCTTCACTCCATCTGTAGA ACAATGCAAGGAAAGCATTGAACAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGTT GTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTGACTCCTCACCATGTGCTGGTTGATGAATACACAGGAGAATGGGTTGATTCACAGTTCATCAACGGAAAATGCAGCAATTACATATGCCCCACTGTCCATAACTCTACAACCTGGCATTCTGACTATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCATGGACATCACCTTCTTCTCAGAGGACGGAGAGCTATCATCC CTGGGAAAGGAGGGCACAGGGTTCAGAAGTAACTACTTTGCTTATGAAACTGGAGGCAAGGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCTCAGACCTCAGTGGATGTAAGTCTAATTCAGGACGTTGAGAGGATCTTGGATTA TTCCCTCTGCCAAGAAACCTGGAGCAAAATCAGAGCGGGTCTTCCAATCTCTCCAGTGGATCTCAGCTATCTTGCTCCTAAAAACCCAGGAACCGGTCCTGCTTTCACCATAATCAATGGTACCCTAAAATACTTTGAGACCAGATACATCAGAGTCGATATTGCTGCTCCAATCCTCT CAAGAATGGTCGGAATGATCAGTGGAACTACCACAGAAAGGGAACTGTGGGATGACTGGGCACCATATGA AGACGTGGAAATTGGACCCAATGGAGTTCTGAGGACCAGTTCAGGATATAAGTTTCCTTTATACATGATTGGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTCAAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTATTTTTTGGTGATACTGGGCTATCCAAAAATCCAATCGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTTCTCCGAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAAAGACAGAT TTATACAGACATAGAGATGAGAATTC 38Rev; RSV ATGGCAGGAAGAAGCGGAGACAGCGACGAAGAAC promoter;TCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATC TranscriptionAAAGCAACCCACCTCCCAATCCCGAGGGGACCCGA CAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGG ATCCTTAGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACG CAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAATAG 39 Rev; HIV Rev;ATGGCAGGAAGAAGCGGAGACAGCGACGAAGAAC Nuclear exportTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATC and stabilizeAAAGCAACCCACCTCCCAATCCCGAGGGGACCCGA viral mRNACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAG AGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTAGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACG CAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAATAG 40 RSV promoterCAATTGCGATGTACGGGCCAGATATACGCGTATCTG and HIV RevAGGGGACTAGGGTGTGTTTAGGCGAAAAGCGGGGC TTCGGTTGTACGCGGTTAGGAGTCCCCTCAGGATATAGTAGTTTCGCTTTTGCATAGGGAGGGGGAAATGTAGTCTTATGCAATACACTTGTAGTCTTGCAACATGGT AACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGG TGGTACGATCGTGCCTTATTAGGAAGGCAACAGACAGGTCTGACATGGATTGGACGAACCACTGAATTCCGCATTGCAGAGATAATTGTATTTAAGTGCCTAGCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTCGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAAGCTAGCGATTAGGCATCTCCTATGGCAGGA AGAAGCGGAGACAGCGACGAAGAACTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACCC ACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAG ACAGATCCATTCGATTAGTGAACGGATCCTTAGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAA CGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAATAT TGGAGTCAGGAGCTAAAGAATAGTCTAGA 41Elongation CCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGG Factor-1 alphaGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCC (EF1-alpha)GAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGT promoterCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGC CAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGG GGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGG GGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCG GCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGA GCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTT TCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAA AGTTTTTTTCTTCCATTTCAGGTGTCGTGA 42Promoter; PGK GGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTC CGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGT TCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAG GGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGA GCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGA CCTCTCTCCCCAG 43 Promoter; UbCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCT CCTCACGGCGAGCGCTGCCACGTCAGACGAAGGGCGCAGGAGCGTTCCTGATCCTTCCGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAG AACCCCAGTATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCA GAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTG AACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGGTGAGTTGCGGGCTGCTGGGCTGGCCGGGGCTTTCGT GGCCGCCGGGCCGCTCGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAGGGCTGTAGTCTGGGTCCGCGA GCAAGGTTGCCCTGAACTGGGGGTTGGGGGGAGCGCACAAAATGGCGGCTGTTCCCGAGTCTTGAATGGAA GACGCTTGTAAGGCGGGCTGTGAGGTCGTTGAAACAAGGTGGGGGGCATGGTGGGCGGCAAGAACCCAAG GTCTTGAGGCCTTCGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGGCTGGGGCACCATCTGGGGACC CTGACGTGAAGTTTGTCACTGACTGGAGAACTCGGGTTTGTCGTCTGGTTGCGGGGGCGGCAGTTATGCGGTGCCGTTGGGCAGTGCACCCGTACCTTTGGGAGCGCGCGCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAATGCAGGGTGGGGCCACCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGACGCAGGGTT CGGGCCTAGGGTAGGCTCTCCTGAATCGACAGGCGCCGGACCTCTGGTGAGGGGAGGGATAAGTGAGGCGTCAGTTTCTTTGGTCGGTTTTATGTACCTATCTTCTTAAGTAGCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTGTTTTGTGAAGTTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATATGTAAT TTTCAGTGTTAGACTAGTAAA 44Poly A; SV40 GTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAA TGTATCTTATCA 45 Poly A; bGHGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTG GGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCG GTGGGCTCTATGG 46 Envelope;ATGAAACTCCCAACAGGAATGGTCATTTTATGTAGC RD114CTAATAATAGTTCGGGCAGGGTTTGACGACCCCCGC AAGGCTATCGCATTAGTACAAAAACAACATGGTAAACCATGCGAATGCAGCGGAGGGCAGGTATCCGAGG CCCCACCGAACTCCATCCAACAGGTAACTTGCCCAGGCAAGACGGCCTACTTAATGACCAACCAAAAATGG AAATGCAGAGTCACTCCAAAAAATCTCACCCCTAGCGGGGGAGAACTCCAGAACTGCCCCTGTAACACTTTCCAGGACTCGATGCACAGTTCTTGTTATACTGAATAC CGGCAATGCAGGGCGAATAATAAGACATACTACACGGCCACCTTGCTTAAAATACGGTCTGGGAGCCTCAACGAGGTACAGATATTACAAAACCCCAATCAGCTCCTACAGTCCCCTTGTAGGGGCTCTATAAATCAGCCCGTTTGCTGGAGTGCCACAGCCCCCATCCATATCTCCGA TGGTGGAGGACCCCTCGATACTAAGAGAGTGTGGACAGTCCAAAAAAGGCTAGAACAAATTCATAAGGCT ATGCATCCTGAACTTCAATACCACCCCTTAGCCCTGCCCAAAGTCAGAGATGACCTTAGCCTTGATGCACGGACTTTTGATATCCTGAATACCACTTTTAGGTTACTCCAGATGTCCAATTTTAGCCTTGCCCAAGATTGTTGGCTCTGTTTAAAACTAGGTACCCCTACCCCTCTTGCGATACCCACTCCCTCTTTAACCTACTCCCTAGCAGACTCCCTAGCGAATGCCTCCTGTCAGATTATACCTCCCCTCTTGGTTCAACCGATGCAGTTCTCCAACTCGTCCTGTTTATCTTCCCCTTTCATTAACGATACGGAACAAATAGACTTAGGTGCAGTCACCTTTACTAACTGCACCTCTGTAGCCAATGTCAGTAGTCCTTTATGTGCCCTAAACGGGTCAGTCTTCCTCTGTGGAAATAACATGGCATACACCTATTTACCCCAAAACTGGACAGGACTTTGCGTCCAAGCCTCCCTCCTCCCCGACATTGACATCATCCCGGGGGATGAGCCAGTCCCCATTCCTGCCATTGATCATTATATACATAGACCTAAACGAGCTGTACAGTTCATCCCTTTACTAGCTGGACTGGGAATCACCGCAGCATTCACCACCGGAGCTACAGGCCTAGGTGTCTCCGTCACCCAGTATACAAAATTATCCCATCAGTTAATATCTGATGTCCAAGTCTTATCCGGTACCATACAAGATTTACAAGACCAGGTAGACTCGTTAGCTGAAGTAGTTCTCCA AAATAGGAGGGGACTGGACCTACTAACGGCAGAACAAGGAGGAATTTGTTTAGCCTTACAAGAAAAATGCTGTTTTTATGCTAACAAGTCAGGAATTGTGAGAAACA AAATAAGAACCCTACAAGAAGAATTACAAAAACGCAGGGAAAGCCTGGCATCCAACCCTCTCTGGACCGGGCTGCAGGGCTTTCTTCCGTACCTCCTACCTCTCCTGGGACCCCTACTCACCCTCCTACTCATACTAACCATTGGGCCATGCGTTTTCAATCGATTGGTCCAATTTGTTAAAGACAGGATCTCAGTGGTCCAGGCTCTGGTTTTG ACTCAGCAATATCACCAGCTAAAACCCATAGAGTACGAGCCATGA 47 Envelope; ATGCTTCTCACCTCAAGCCCGCACCACCTTCGGCAC GALVCAGATGAGTCCTGGGAGCTGGAAAAGACTGATCAT CCTCTTAAGCTGCGTATTCGGAGACGGCAAAACGAGTCTGCAGAATAAGAACCCCCACCAGCCTGTGACCCTCACCTGGCAGGTACTGTCCCAAACTGGGGACGTTGTCTGGGACAAAAAGGCAGTCCAGCCCCTTTGGACTTGGTGGCCCTCTCTTACACCTGATGTATGTGCCCTGGCGGCCGGTCTTGAGTCCTGGGATATCCCGGGATCCGATGTATCGTCCTCTAAAAGAGTTAGACCTCCTGATTCAGACTATACTGCCGCTTATAAGCAAATCACCTGGG GAGCCATAGGGTGCAGCTACCCTCGGGCTAGGACCAGGATGGCAAATTCCCCCTTCTACGTGTGTCCCCGA GCTGGCCGAACCCATTCAGAAGCTAGGAGGTGTGGGGGGCTAGAATCCCTATACTGTAAAGAATGGAGTT GTGAGACCACGGGTACCGTTTATTGGCAACCCAAGTCCTCATGGGACCTCATAACTGTAAAATGGGACCAA AATGTGAAATGGGAGCAAAAATTTCAAAAGTGTGAACAAACCGGCTGGTGTAACCCCCTCAAGATAGACTT CACAGAAAAAGGAAAACTCTCCAGAGATTGGATAACGGAAAAAACCTGGGAATTAAGGTTCTATGTATATGGACACCCAGGCATACAGTTGACTATCCGCTTAGAGGTCACTAACATGCCGGTTGTGGCAGTGGGCCCAGACCCTGTCCTTGCGGAACAGGGACCTCCTAGCAAGCCCCTCACTCTCCCTCTCTCCCCACGGAAAGCGCCGCCCACCCCTCTACCCCCGGCGGCTAGTGAGCAAACCCCTGCGGTGCATGGAGAAACTGTTACCCTAAACTCTCCGCCTCCCACCAGTGGCGACCGACTCTTTGGCCTTGTGCAGGGGGCCTTCCTAACCTTGAATGCTACCAACCCAGGGGCCACTAAGTCTTGCTGGCTCTGTTTGGGCATGAGCCCCCCTTATTATGAAGGGATAGCCTCTTCAGGAGAGGTCGCTTATACCTCCAACCATACCCGATGCCACT GGGGGGCCCAAGGAAAGCTTACCCTCACTGAGGTCTCCGGACTCGGGTCATGCATAGGGAAGGTGCCTCTTACCCATCAACATCTTTGCAACCAGACCTTACCCATCAATTCCTCTAAAAACCATCAGTATCTGCTCCCCTCAAACCATAGCTGGTGGGCCTGCAGCACTGGCCTCACCCCCTGCCTCTCCACCTCAGTTTTTAATCAGTCTAAAGACTTCTGTGTCCAGGTCCAGCTGATCCCCCGCATCTATTACCATTCTGAAGAAACCTTGTTACAAGCCTATGACAAATCACCCCCCAGGTTTAAAAGAGAGCCTGCCTCACTTACCCTAGCTGTCTTCCTGGGGTTAGGGATTGCGGCAGGTATAGGTACTGGCTCAACCGCCCTAATTA AAGGGCCCATAGACCTCCAGCAAGGCCTAACCAGCCTCCAAATCGCCATTGACGCTGACCTCCGGGCCCTT CAGGACTCAATCAGCAAGCTAGAGGACTCACTGACTTCCCTATCTGAGGTAGTACTCCAAAATAGGAGAGGCCTTGACTTACTATTCCTTAAAGAAGGAGGCCTCTGCGCGGCCCTAAAAGAAGAGTGCTGTTTTTATGTAGA CCACTCAGGTGCAGTACGAGACTCCATGAAAAAACTTAAAGAAAGACTAGATAAAAGACAGTTAGAGCGC CAGAAAAACCAAAACTGGTATGAAGGGTGGTTCAATAACTCCCCTTGGTTTACTACCCTACTATCAACCATCGCTGGGCCCCTATTGCTCCTCCTTTTGTTACTCACTCTTGGGCCCTGCATCATCAATAAATTAATCCAATTCATCAATGATAGGATAAGTGCAGTCAAAATTTTAGTCC TTAGACAGAAATATCAGACCCTAGATAACGAGGAAAACCTTTAA 48 Envelope; FUG ATGGTTCCGCAGGTTCTTTTGTTTGTACTCCTTCTGGGTTTTTCGTTGTGTTTCGGGAAGTTCCCCATTTACACGATACCAGACGAACTTGGTCCCTGGAGCCCTATTGACATACACCATCTCAGCTGTCCAAATAACCTGGTTGTGGAGGATGAAGGATGTACCAACCTGTCCGAGTTCTCCTACATGGAACTCAAAGTGGGATACATCTCAGCCATCAAAGTGAACGGGTTCACTTGCACAGGTGTTGTGACAGAGGCAGAGACCTACACCAACTTTGTTGGTTATGTCACAACCACATTCAAGAGAAAGCATTTCCGCCCCAC CCCAGACGCATGTAGAGCCGCGTATAACTGGAAGATGGCCGGTGACCCCAGATATGAAGAGTCCCTACAC AATCCATACCCCGACTACCACTGGCTTCGAACTGTAAGAACCACCAAAGAGTCCCTCATTATCATATCCCCAAGTGTGACAGATTTGGACCCATATGACAAATCCCTTCACTCAAGGGTCTTCCCTGGCGGAAAGTGCTCAGGAATAACGGTGTCCTCTACCTACTGCTCAACTAACCATGATTACACCATTTGGATGCCCGAGAATCCGAGACCAAGGACACCTTGTGACATTTTTACCAATAGCAGAGGG AAGAGAGCATCCAACGGGAACAAGACTTGCGGCTTTGTGGATGAAAGAGGCCTGTATAAGTCTCTAAAAG GAGCATGCAGGCTCAAGTTATGTGGAGTTCTTGGACTTAGACTTATGGATGGAACATGGGTCGCGATGCAA ACATCAGATGAGACCAAATGGTGCCCTCCAGATCAGTTGGTGAATTTGCACGACTTTCGCTCAGACGAGAT CGAGCATCTCGTTGTGGAGGAGTTAGTTAAGAAAAGAGAGGAATGTCTGGATGCATTAGAGTCCATCATG ACCACCAAGTCAGTAAGTTTCAGACGTCTCAGTCACCTGAGAAAACTTGTCCCAGGGTTTGGAAAAGCATATACCATATTCAACAAAACCTTGATGGAGGCTGATGCTCACTACAAGTCAGTCCGGACCTGGAATGAGATCATC CCCTCAAAAGGGTGTTTGAAAGTTGGAGGAAGGTGCCATCCTCATGTGAACGGGGTGTTTTTCAATGGTATAATATTAGGGCCTGACGACCATGTCCTAATCCCAGAGATGCAATCATCCCTCCTCCAGCAACATATGGAGTTGTTGGAATCTTCAGTTATCCCCCTGATGCACCCCCTGGCAGACCCTTCTACAGTTTTCAAAGAAGGTGATGAGGCTGAGGATTTTGTTGAAGTTCACCTCCCCGATGTGTACAAACAGATCTCAGGGGTTGACCTGGGTCTCCC GAACTGGGGAAAGTATGTATTGATGACTGCAGGGGCCATGATTGGCCTGGTGTTGATATTTTCCCTAATGACATGGTGCAGAGTTGGTATCCATCTTTGCATTAAAT TAAAGCACACCAAGAAAAGACAGATTTATACAGACATAGAGATGAACCGACTTGGAAAGTAA 49 Envelope;ATGGGTCAGATTGTGACAATGTTTGAGGCTCTGCCT LCMVCACATCATCGATGAGGTGATCAACATTGTCATTATTGTGCTTATCGTGATCACGGGTATCAAGGCTGTCTACAATTTTGCCACCTGTGGGATATTCGCATTGATCAGTTTCCTACTTCTGGCTGGCAGGTCCTGTGGCATGTACGGTCTTAAGGGACCCGACATTTACAAAGGAGTTTACCAATTTAAGTCAGTGGAGTTTGATATGTCACATCTGAACCTGACCATGCCCAACGCATGTTCAGCCAACAACTCCCACCATTACATCAGTATGGGGACTTCTGGACTAGAATTGACCTTCACCAATGATTCCATCATCAGTCACAACTTTTGCAATCTGACCTCTGCCTTCAACAAAAAGACCTTTGACCACACACTCATGAGTATAGTTTCGAGCCTACACCTCAGTATCAGAGGGAACTCCAACTATAAGGCAGTATCCTGCGACTTCAACAATGGCATAACCATCCAATACAACTTGACATTCTCAGATCGACAAAGTGCT CAGAGCCAGTGTAGAACCTTCAGAGGTAGAGTCCTAGATATGTTTAGAACTGCCTTCGGGGGGAAATACAT GAGGAGTGGCTGGGGCTGGACAGGCTCAGATGGCAAGACCACCTGGTGTAGCCAGACGAGTTACCAATAC CTGATTATACAAAATAGAACCTGGGAAAACCACTGCACATATGCAGGTCCTTTTGGGATGTCCAGGATTCTCCTTTCCCAAGAGAAGACTAAGTTCTTCACTAGGAGACTAGCGGGCACATTCACCTGGACTTTGTCAGACTCTTCAGGGGTGGAGAATCCAGGTGGTTATTGCCTGACCAAATGGATGATTCTTGCTGCAGAGCTTAAGTGTTT CGGGAACACAGCAGTTGCGAAATGCAATGTAAATCATGATGCCGAATTCTGTGACATGCTGCGACTAATTG ACTACAACAAGGCTGCTTTGAGTAAGTTCAAAGAGGACGTAGAATCTGCCTTGCACTTATTCAAAACAACAGTGAATTCTTTGATTTCAGATCAACTACTGATGAGGAACCACTTGAGAGATCTGATGGGGGTGCCATATTGCAATTACTCAAAGTTTTGGTACCTAGAACATGCAAAGACCGGCGAAACTAGTGTCCCCAAGTGCTGGCTTGTCACCAATGGTTCTTACTTAAATGAGACCCACTTCAGT GATCAAATCGAACAGGAAGCCGATAACATGATTACAGAGATGTTGAGGAAGGATTACATAAAGAGGCAGG GGAGTACCCCCCTAGCATTGATGGACCTTCTGATGTTTTCCACATCTGCATATCTAGTCAGCATCTTCCTGCACCTTGTCAAAATACCAACACACAGGCACATAAAAG GTGGCTCATGTCCAAAGCCACACCGATTAACCAACAAAGGAATTTGTAGTTGTGGTGCATTTAAGGTGCCTG GTGTAAAAACCGTCTGGAAAAGACGCTGA 50Envelope; FPV ATGAACACTCAAATCCTGGTTTTCGCCCTTGTGGCAGTCATCCCCACAAATGCAGACAAAATTTGTCTTGGA CATCATGCTGTATCAAATGGCACCAAAGTAAACACACTCACTGAGAGAGGAGTAGAAGTTGTCAATGCAA CGGAAACAGTGGAGCGGACAAACATCCCCAAAATTTGCTCAAAAGGGAAAAGAACCACTGATCTTGGCCA ATGCGGACTGTTAGGGACCATTACCGGACCACCTCAATGCGACCAATTTCTAGAATTTTCAGCTGATCTAAT AATCGAGAGACGAGAAGGAAATGATGTTTGTTACCCGGGGAAGTTTGTTAATGAAGAGGCATTGCGACAA ATCCTCAGAGGATCAGGTGGGATTGACAAAGAAACAATGGGATTCACATATAGTGGAATAAGGACCAACG GAACAACTAGTGCATGTAGAAGATCAGGGTCTTCATTCTATGCAGAAATGGAGTGGCTCCTGTCAAATACAGACAATGCTGCTTTCCCACAAATGACAAAATCATACA AAAACACAAGGAGAGAATCAGCTCTGATAGTCTGGGGAATCCACCATTCAGGATCAACCACCGAACAGAC CAAACTATATGGGAGTGGAAATAAACTGATAACAGTCGGGAGTTCCAAATATCATCAATCTTTTGTGCCGA GTCCAGGAACACGACCGCAGATAAATGGCCAGTCCGGACGGATTGATTTTCATTGGTTGATCTTGGATCCCAATGATACAGTTACTTTTAGTTTCAATGGGGCTTTCATAGCTCCAAATCGTGCCAGCTTCTTGAGGGGAAAGTCCATGGGGATCCAGAGCGATGTGCAGGTTGATGCC AATTGCGAAGGGGAATGCTACCACAGTGGAGGGACTATAACAAGCAGATTGCCTTTTCAAAACATCAATAG CAGAGCAGTTGGCAAATGCCCAAGATATGTAAAACAGGAAAGTTTATTATTGGCAACTGGGATGAAGAAC GTTCCCGAACCTTCCAAAAAAAGGAAAAAAAGAGGCCTGTTTGGCGCTATAGCAGGGTTTATTGAAAATGGTTGGGAAGGTCTGGTCGACGGGTGGTACGGTTTCAG GCATCAGAATGCACAAGGAGAAGGAACTGCAGCAGACTACAAAAGCACCCAATCGGCAATTGATCAGATA ACCGGAAAGTTAAATAGACTCATTGAGAAAACCAACCAGCAATTTGAGCTAATAGATAATGAATTCACTGA GGTGGAAAAGCAGATTGGCAATTTAATTAACTGGACCAAAGACTCCATCACAGAAGTATGGTCTTACAATGCTGAACTTCTTGTGGCAATGGAAAACCAGCACACTATTGATTTGGCTGATTCAGAGATGAACAAGCTGTATG AGCGAGTGAGGAAACAATTAAGGGAAAATGCTGAAGAGGATGGCACTGGTTGCTTTGAAATTTTTCATAAATGTGACGATGATTGTATGGCTAGTATAAGGAACAAT ACTTATGATCACAGCAAATACAGAGAAGAAGCGATGCAAAATAGAATACAAATTGACCCAGTCAAATTGA GTAGTGGCTACAAAGATGTGATACTTTGGTTTAGCTTCGGGGCATCATGCTTTTTGCTTCTTGCCATTGCAATGGGCCTTGTTTTCATATGTGTGAAGAACGGAAACAT GCGGTGCACTATTTGTATATAA 51Envelope; RRV AGTGTAACAGAGCACTTTAATGTGTATAAGGCTACTAGACCATACCTAGCACATTGCGCCGATTGCGGGGA CGGGTACTTCTGCTATAGCCCAGTTGCTATCGAGGAGATCCGAGATGAGGCGTCTGATGGCATGCTTAAGAT CCAAGTCTCCGCCCAAATAGGTCTGGACAAGGCAGGCACCCACGCCCACACGAAGCTCCGATATATGGCTGGTCATGATGTTCAGGAATCTAAGAGAGATTCCTTGAGGGTGTACACGTCCGCAGCGTGCTCCATACATGGGACGATGGGACACTTCATCGTCGCACACTGTCCACCAGGCGACTACCTCAAGGTTTCGTTCGAGGACGCAGATT CGCACGTGAAGGCATGTAAGGTCCAATACAAGCACAATCCATTGCCGGTGGGTAGAGAGAAGTTCGTGGTTAGACCACACTTTGGCGTAGAGCTGCCATGCACCTCA TACCAGCTGACAACGGCTCCCACCGACGAGGAGATTGACATGCATACACCGCCAGATATACCGGATCGCAC CCTGCTATCACAGACGGCGGGCAACGTCAAAATAACAGCAGGCGGCAGGACTATCAGGTACAACTGTACC TGCGGCCGTGACAACGTAGGCACTACCAGTACTGACAAGACCATCAACACATGCAAGATTGACCAATGCC ATGCTGCCGTCACCAGCCATGACAAATGGCAATTTACCTCTCCATTTGTTCCCAGGGCTGATCAGACAGCTAGGAAAGGCAAGGTACACGTTCCGTTCCCTCTGACTAACGTCACCTGCCGAGTGCCGTTGGCTCGAGCGCCGG ATGCCACCTATGGTAAGAAGGAGGTGACCCTGAGATTACACCCAGATCATCCGACGCTCTTCTCCTATAGG AGTTTAGGAGCCGAACCGCACCCGTACGAGGAATGGGTTGACAAGTTCTCTGAGCGCATCATCCCAGTGAC GGAAGAAGGGATTGAGTACCAGTGGGGCAACAACCCGCCGGTCTGCCTGTGGGCGCAACTGACGACCGAG GGCAAACCCCATGGCTGGCCACATGAAATCATTCAGTACTATTATGGACTATACCCCGCCGCCACTATTGCCGCAGTATCCGGGGCGAGTCTGATGGCCCTCCTAACTCTGGCGGCCACATGCTGCATGCTGGCCACCGCGAG GAGAAAGTGCCTAACACCGTACGCCCTGACGCCAGGAGCGGTGGTACCGTTGACACTGGGGCTGCTTTGCT GCGCACCGAGGGCGAATGCA 52Envelope; MLV AGTGTAACAGAGCACTTTAATGTGTATAAGGCTACT 10A1AGACCATACCTAGCACATTGCGCCGATTGCGGGGA CGGGTACTTCTGCTATAGCCCAGTTGCTATCGAGGAGATCCGAGATGAGGCGTCTGATGGCATGCTTAAGAT CCAAGTCTCCGCCCAAATAGGTCTGGACAAGGCAGGCACCCACGCCCACACGAAGCTCCGATATATGGCTGGTCATGATGTTCAGGAATCTAAGAGAGATTCCTTGAGGGTGTACACGTCCGCAGCGTGCTCCATACATGGGACGATGGGACACTTCATCGTCGCACACTGTCCACCAGGCGACTACCTCAAGGTTTCGTTCGAGGACGCAGATT CGCACGTGAAGGCATGTAAGGTCCAATACAAGCACAATCCATTGCCGGTGGGTAGAGAGAAGTTCGTGGTTAGACCACACTTTGGCGTAGAGCTGCCATGCACCTCA TACCAGCTGACAACGGCTCCCACCGACGAGGAGATTGACATGCATACACCGCCAGATATACCGGATCGCAC CCTGCTATCACAGACGGCGGGCAACGTCAAAATAACAGCAGGCGGCAGGACTATCAGGTACAACTGTACC TGCGGCCGTGACAACGTAGGCACTACCAGTACTGACAAGACCATCAACACATGCAAGATTGACCAATGCC ATGCTGCCGTCACCAGCCATGACAAATGGCAATTTACCTCTCCATTTGTTCCCAGGGCTGATCAGACAGCTAGGAAAGGCAAGGTACACGTTCCGTTCCCTCTGACTAACGTCACCTGCCGAGTGCCGTTGGCTCGAGCGCCGG ATGCCACCTATGGTAAGAAGGAGGTGACCCTGAGATTACACCCAGATCATCCGACGCTCTTCTCCTATAGG AGTTTAGGAGCCGAACCGCACCCGTACGAGGAATGGGTTGACAAGTTCTCTGAGCGCATCATCCCAGTGAC GGAAGAAGGGATTGAGTACCAGTGGGGCAACAACCCGCCGGTCTGCCTGTGGGCGCAACTGACGACCGAG GGCAAACCCCATGGCTGGCCACATGAAATCATTCAGTACTATTATGGACTATACCCCGCCGCCACTATTGCCGCAGTATCCGGGGCGAGTCTGATGGCCCTCCTAACTCTGGCGGCCACATGCTGCATGCTGGCCACCGCGAG GAGAAAGTGCCTAACACCGTACGCCCTGACGCCAGGAGCGGTGGTACCGTTGACACTGGGGCTGCTTTGCT GCGCACCGAGGGCGAATGCA 53Envelope; Ebola ATGGGTGTTACAGGAATATTGCAGTTACCTCGTGATCGATTCAAGAGGACATCATTCTTTCTTTGGGTAATTATCCTTTTCCAAAGAACATTTTCCATCCCACTTGGAGTCATCCACAATAGCACATTACAGGTTAGTGATGTCGACAAACTGGTTTGCCGTGACAAACTGTCATCCACA AATCAATTGAGATCAGTTGGACTGAATCTCGAAGGGAATGGAGTGGCAACTGACGTGCCATCTGCAACTA AAAGATGGGGCTTCAGGTCCGGTGTCCCACCAAAGGTGGTCAATTATGAAGCTGGTGAATGGGCTGAAAA CTGCTACAATCTTGAAATCAAAAAACCTGACGGGAGTGAGTGTCTACCAGCAGCGCCAGACGGGATTCGG GGCTTCCCCCGGTGCCGGTATGTGCACAAAGTATCAGGAACGGGACCGTGTGCCGGAGACTTTGCCTTCCACAAAGAGGGTGCTTTCTTCCTGTATGACCGACTTGCTTCCACAGTTATCTACCGAGGAACGACTTTCGCTGAAGGTGTCGTTGCATTTCTGATACTGCCCCAAGCTAAGAAGGACTTCTTCAGCTCACACCCCTTGAGAGAGCCGGTCAATGCAACGGAGGACCCGTCTAGTGGCTACTATTCTACCACAATTAGATATCAAGCTACCGGTTTTGGAACCAATGAGACAGAGTATTTGTTCGAGGTTGACAATTTGACCTACGTCCAACTTGAATCAAGATTCACACCACAGTTTCTGCTCCAGCTGAATGAGACAATATATACA AGTGGGAAAAGGAGCAATACCACGGGAAAACTAATTTGGAAGGTCAACCCCGAAATTGATACAACAATCG GGGAGTGGGCCTTCTGGGAAACTAAAAAAACCTCACTAGAAAAATTCGCAGTGAAGAGTTGTCTTTCACAG CTGTATCAAACAGAGCCAAAAACATCAGTGGTCAGAGTCCGGCGCGAACTTCTTCCGACCCAGGGACCAAC ACAACAACTGAAGACCACAAAATCATGGCTTCAGAAAATTCCTCTGCAATGGTTCAAGTGCACAGTCAAGGAAGGGAAGCTGCAGTGTCGCATCTGACAACCCTTGCCACAATCTCCACGAGTCCTCAACCCCCCACAACCAA ACCAGGTCCGGACAACAGCACCCACAATACACCCGTGTATAAACTTGACATCTCTGAGGCAACTCAAGTTG AACAACATCACCGCAGAACAGACAACGACAGCACAGCCTCCGACACTCCCCCCGCCACGACCGCAGCCGGA CCCCTAAAAGCAGAGAACACCAACACGAGCAAGGGTACCGACCTCCTGGACCCCGCCACCACAACAAGTCC CCAAAACCACAGCGAGACCGCTGGCAACAACAACACTCATCACCAAGATACCGGAGAAGAGAGTGCCAGC AGCGGGAAGCTAGGCTTAATTACCAATACTATTGCTGGAGTCGCAGGACTGATCACAGGCGGGAGGAGAGC TCGAAGAGAAGCAATTGTCAATGCTCAACCCAAATGCAACCCTAATTTACATTACTGGACTACTCAGGATGAAGGTGCTGCAATCGGACTGGCCTGGATACCATATT TCGGGCCAGCAGCCGAGGGAATTTACATAGAGGGGCTGATGCACAATCAAGATGGTTTAATCTGTGGGTTG AGACAGCTGGCCAACGAGACGACTCAAGCTCTTCAACTGTTCCTGAGAGCCACAACCGAGCTACGCACCTTTTCAATCCTCAACCGTAAGGCAATTGATTTCTTGCT GCAGCGATGGGGCGGCACATGCCACATTTTGGGACCGGACTGCTGTATCGAACCACATGATTGGACCAAG AACATAACAGACAAAATTGATCAGATTATTCATGATTTTGTTGATAAAACCCTTCCGGACCAGGGGGACAAT GACAATTGGTGGACAGGATGGAGACAATGGATACCGGCAGGTATTGGAGTTACAGGCGTTATAATTGCAGTTATCGCTTTATTCTGTATATGCAAATTTGTCTTTTAG 54 Polymerase IIITTTCCCATGATTCCTTCATATTTGCATATACGATACA shRNAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTG promoters; U6TAAACACAAAGATATTAGTACAAAATACGTGACGT promoterAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATA TCTTGTGGAAAGGACGAAAC 55Polymerase III CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGC shRNAATTCTGGATAGTGTCAAAACAGCCGGAAATCAAGT promoters; 7SKCCGTTTATCTCAAACTTTAGCATTTTGGGAATAAAT promoterGATATTTGCTATGCTGGTTAAATTAGATTTTAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGCAACTTGACCTAAGTGTAAAGTTGAGATTTCCTTCAGGTTTA TATAGCTTGTGCGCCGCCTGGCTACCTC 56FDPS target GTCCTGGAGTACAATGCCATT sequence #1 57 FDPS targetGCAGGATTTCGTTCAGCACTT sequence #2 58 FDPS target GCCATGTACATGGCAGGAATTsequence #3 59 FDPS target GCAGAAGGAGGCTGAGAAAGT sequence #4 60Non-targeting GCCGCTTTGTAGGATAGAGCTCGAGCTCTATCCTAC sequenceAAAGCGGCTTTTT 61 Forward primer AGGAATTGATGGCGAGAAGG 62 Reverse primerCCCAAAGAGGTCAAGGTAATCA 63 Forward primer AGCGCGGCTACAGCTTCA 64Reverse primer GGCGACGTAGCACAGCTTCT 65 Left InvertedCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGG Terminal RepeatCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCC (Left ITR)TCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGC CAACTCCATCACTAGGGGTTCCT 66Right Inverted GAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGC Terminal RepeatCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGC (Right ITR)CGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTG CCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention.

What is claimed is:
 1. An immunotherapy-based system comprising: (i) atleast one helper plasmid comprising DNA sequences for expressing afunctional protein derived from each of a gag, pol, and rev gene; (ii)an envelope plasmid comprising a DNA sequence for expressing an envelopeprotein capable of infecting a target cell; and (iii) a therapeuticvector comprising: at least one encoded shRNA that, when expressed,inhibits production of farnesyl diphosphate synthase, or, at least oneencoded microRNA that, when expressed, inhibits production of farnesyldiphosphate synthase.
 2. The immunotherapy-based system of claim 1,wherein the at least one helper plasmid comprises first and secondhelper plasmids, wherein the first help helper plasmid comprises DNAsequences for expressing such proteins derived from the gag and polgenes, and the second helper plasmid comprises a DNA sequence forexpressing such protein derived from the rev gene.
 3. Theimmunotherapy-based system of claim 1, wherein the target cell comprisesa cancer cell that is present in a cancer selected from one or more of acarcinoma, a leukemia, a lymphoma, a sarcoma, a myeloma, a mesothelioma,a mixed type, or mixtures thereof.
 4. The immunotherapy-based system ofclaim 1, further comprising an aminobisphosphonate drug.
 5. Theimmunotherapy-based system of claim 4, wherein the aminobisphosphonatedrug comprises zoledronic acid.
 6. The immunotherapy-based system ofclaim 1, wherein the at least one encoded shRNA comprises a sequencehaving at least 80 percent identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, or SEQ ID NO: 4, or wherein the at least one encoded microRNAcomprises a sequence having at least 80 percent identity with SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:10.